Excipient compounds for protein processing

ABSTRACT

Disclosed herein are methods for improving a parameter of a protein-related process comprising providing a viscosity-reducing excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short-chain organic acids, and low molecular weight aliphatic polyacids, and adding a viscosity-reducing amount of the viscosity-reducing excipient compound to a carrier solution for the protein-related process, wherein the carrier solution contains a protein of interest, and carrier solutions comprising a liquid medium in which is dissolved a protein of interest, and a viscosity-reducing excipient, wherein the viscosity of the carrier solution has a lower viscosity that that of a control solution that is substantially similar to the carrier solution except for the presence of the viscosity-reducing excipient.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/896,374 filed Feb. 14, 2018, which claims the benefit of U.S.Provisional Application No. 62/459,893 filed Feb. 16, 2017; U.S.application Ser. No. 15/896,374 is also a continuation-in-part of U.S.application Ser. No. 15/331,197 filed Oct. 21, 2016 (now U.S. Pat. No.10,478,498), which is a continuation-in-part of U.S. application Ser.No. 14/966,549 filed Dec. 11, 2015 (now U.S. Pat. No. 9,605,051), whichis a continuation of U.S. application Ser. No. 14/744,847 filed Jun. 19,2015 (Abandoned), which claims the benefit of U.S. ProvisionalApplication No. 62/014,784 filed Jun. 20, 2014, U.S. ProvisionalApplication No. 62/083,623, filed Nov. 24, 2014, and U.S. ProvisionalApplication Ser. No. 62/136,763 filed Mar. 23, 2015; U.S. applicationSer. No. 14/966,549 also claims the benefit of U.S. ProvisionalApplication No. 62/245,513, filed Oct. 23, 2015; U.S. application Ser.No. 15/331,197 also claims the benefit of U.S. Provisional ApplicationNo. 62/245,513, filed Oct. 23, 2015. The entire contents of the each ofthe above applications are incorporated by reference herein.

FIELD OF APPLICATION

This application relates generally to formulations for delivering andprocessing biopolymers.

BACKGROUND

Biopolymers may be used for therapeutic or non-therapeutic purposes.Biopolymer-based therapeutics, such as antibody or enzyme formulations,are widely used in treating disease. Non-therapeutic biopolymers, suchas enzymes, peptides, and structural proteins, have utility innon-therapeutic applications such as household, nutrition, commercial,and industrial uses.

Biopolymers used in therapeutic applications must be formulated topermit their introduction into the body for treatment of disease. Forexample, it is advantageous to deliver antibody and protein/peptidebiopolymer formulations by subcutaneous (SC) or intramuscular (IM)routes under certain circumstances, instead of administering theseformulations by intravenous (IV) injections. In order to achieve betterpatient compliance and comfort with SC or IM injection though, theliquid volume in the syringe is typically limited to 2 to 3 ccs and theviscosity of the formulation is typically lower than about 20 centipoise(cP) so that the formulation can be delivered using conventional medicaldevices and small-bore needles. These delivery parameters do not alwaysfit well with the dosage requirements for the formulations beingdelivered.

Antibodies, for example, may need to be delivered at high dose levels toexert their intended therapeutic effect. Using a restricted liquidvolume to deliver a high dose level of an antibody formulation canrequire a high concentration of the antibody in the delivery vehicle,sometimes exceeding a level of 150 mg/mL. At this dosage level, theviscosity-versus-concentration plots of protein solutions lie beyondtheir linear-nonlinear transition, such that the viscosity of theformulation rises dramatically with increasing concentration. Increasedviscosity, however, is not compatible with standard SC or IM deliverysystems. The solutions of biopolymer-based therapeutics are also proneto stability problems, such as precipitation, hazing, opalescence,denaturing, and gel formation, reversible or irreversible aggregation.The stability problems limit the shelf life of the solutions or requirespecial handling.

One approach to producing protein formulations for injection is totransform the therapeutic protein solution into a powder that can bereconstituted to form a suspension suitable for SC or IM delivery.Lyophilization is a standard technique to produce protein powders.Freeze-drying, spray drying and even precipitation followed bysuper-critical-fluid extraction have been used to generate proteinpowders for subsequent reconstitution. Powdered suspensions are low inviscosity before re-dissolution (compared to solutions at the sameoverall dose) and thus may be suitable for SC or IM injection, providedthe particles are sufficiently small to fit through the needle. However,protein crystals that are present in the powder have the inherent riskof triggering immune response. The uncertain protein stability/activityfollowing re-dissolution poses further concerns. There remains a need inthe art for techniques to produce low viscosity protein formulations fortherapeutic purposes while avoiding the limitations introduced byprotein powder suspensions.

In addition to the therapeutic applications of proteins described above,biopolymers such as enzymes, peptides, and structural proteins can beused in non-therapeutic applications. These non-therapeutic biopolymerscan be produced from a number of different pathways, for example,derived from plant sources, animal sources, or produced from cellcultures.

The non-therapeutic proteins can be produced, transported, stored, andhandled as a granular or powdered material or as a solution ordispersion, usually in water. The biopolymers for non-therapeuticapplications can be globular or fibrous proteins, and certain forms ofthese materials can have limited solubility in water or exhibit highviscosity upon dissolution. These solution properties can presentchallenges to the formulation, handling, storage, pumping, andperformance of the non-therapeutic materials, so there is a need formethods to reduce viscosity and improve solubility and stability ofnon-therapeutic protein solutions.

Proteins are complex biopolymers, each with a uniquely folded 3-Dstructure and surface energy map (hydrophobic/hydrophilic regions andcharges). In concentrated protein solutions, these macromolecules maystrongly interact and even inter-lock in complicated ways, depending ontheir exact shape and surface energy distribution. “Hot-spots” forstrong specific interactions lead to protein clustering, increasingsolution viscosity. To address these concerns, a number of excipientcompounds are used in biotherapeutic formulations, aiming to reducesolution viscosity by impeding localized interactions and clustering.These efforts are individually tailored, often empirically, sometimesguided by in silico simulations. Combinations of excipient compounds maybe provided, but optimizing such combinations again must progressempirically and on a case-by case basis.

There remains a need in the art for a truly universal approach toreducing viscosity in protein formulations at a given concentrationunder nonlinear conditions. There is an additional need in the art toachieve this viscosity reduction while preserving the activity of theprotein. It would be further desirable to adapt the viscosity-reductionsystem to use with formulations having tunable and sustained releaseprofiles, and to use with formulations adapted for depot injection. Inaddition, it is desirable to improve processes for producing proteinsand other biopolymers.

SUMMARY OF THE INVENTION

Disclosed herein, in embodiments, are liquid formulations comprising aprotein and an excipient compound selected from the group consisting ofhindered amines, anionic aromatics, functionalized amino acids,oligopeptides, short-chain organic acids, and low molecular weightaliphatic polyacids, wherein the excipient compound is added in aviscosity-reducing amount. In embodiments, the protein is a PEGylatedprotein and the excipient is a low molecular weight aliphatic polyacid.In embodiments, the formulation is a pharmaceutical composition, and thetherapeutic formulation comprises a therapeutic protein, wherein theexcipient compound is a pharmaceutically acceptable excipient compound.In embodiments, the formulation is a non-therapeutic formulation, andthe non-therapeutic formulation comprises a non-therapeutic protein. Inembodiments, the viscosity-reducing amount reduces viscosity of theformulation to a viscosity less than the viscosity of a controlformulation. In embodiments, the viscosity of the formulation is atleast about 10% less than the viscosity of the control formulation or isat least about 30% less than the viscosity of the control formulation,or is at least about 50% less than the viscosity of the controlformulation, or is at least about 70% less than the viscosity of thecontrol formulation, or is at least about 90% less than the viscosity ofthe control formulation. In embodiments, the viscosity is less thanabout 100 cP, or is less than about 50 cP, or is less than about 20 cP,or is less than about 10 cP. In embodiments, the excipient compound hasa molecular weight of <5000 Da, or <1500 Da, or <500 Da. In embodiments,the formulation contains at least about 25 mg/mL of the protein, or atleast about 100 mg/mL of the protein, or at least about 200 mg/mL of theprotein, or at least about 300 mg/mL of the protein. In embodiments, theformulation comprises between about 5 mg/mL to about 300 mg/mL of theexcipient compound or comprises between about 10 mg/mL to about 200mg/mL of the excipient compound or comprises between about 20 mg/mL toabout 100 mg/mL, or comprises between about 25 mg/mL to about 75 mg/mLof the excipient compound. In embodiments, the formulation has animproved stability when compared to the control formulation. Inembodiments, the excipient compound is a hindered amine. In embodiments,the hindered amine is selected from the group consisting of caffeine,theophylline, tyramine, procaine, lidocaine, imidazole, aspartame,saccharin, and acesulfame potassium. In embodiments, the hindered amineis caffeine. In embodiments, the hindered amine is a local injectableanesthetic compound. The hindered amine can possess an independentpharmacological property, and the hindered amine can be present in theformulation in an amount that has an independent pharmacological effect.In embodiments the hindered amine can be present in the formulation inan amount that is less than a therapeutically effective amount. Theindependent pharmacological activity can be a local anesthetic activity.In embodiments, the hindered amine possessing the independentpharmacological activity is combined with a second excipient compoundthat further decreases the viscosity of the formulation. The secondexcipient compound can be selected from the group consisting ofcaffeine, theophylline, tyramine, procaine, lidocaine, imidazole,aspartame, saccharin, and acesulfame potassium. In embodiments, theformulation can comprise an additional agent selected from the groupconsisting of preservatives, surfactants, sugars, polysaccharides,arginine, proline, hyaluronidase, stabilizers, and buffers.

Further disclosed herein are methods of treating a disease or disorderin a mammal, comprising administering to said mammal a liquidtherapeutic formulation, wherein the therapeutic formulation comprises atherapeutically effective amount of a therapeutic protein, and whereinthe formulation further comprises an pharmaceutically acceptableexcipient compound selected from the group consisting of hinderedamines, anionic aromatics, functionalized amino acids, oligopeptides,short-chain organic acids, and low molecular weight aliphatic polyacids;and wherein the therapeutic formulation is effective for the treatmentof the disease or disorder. In embodiments, the therapeutic protein is aPEGylated protein, and the excipient compound is a low molecular weightaliphatic polyacid. In embodiments, the excipient is a hindered amine.In embodiments, the hindered amine is a local anesthetic compound. Inembodiments, the formulation is administered by subcutaneous injection,or an intramuscular injection, or an intravenous injection. Inembodiments, the excipient compound is present in the therapeuticformulation in a viscosity-reducing amount, and the viscosity-reducingamount reduces viscosity of the therapeutic formulation to a viscosityless than the viscosity of a control formulation. In embodiments, thetherapeutic formulation has an improved stability when compared to thecontrol formulation. In embodiments, the excipient compound isessentially pure.

Further disclosed herein are methods of reducing pain at an injectionsite of a therapeutic protein in a mammal in need thereof, comprising:administering a liquid therapeutic formulation by injection, wherein thetherapeutic formulation comprises a therapeutically effective amount ofthe therapeutic protein, wherein the formulation further comprises anpharmaceutically acceptable excipient compound selected from the groupconsisting of local injectable anesthetic compounds, wherein thepharmaceutically acceptable excipient compound is added to theformulation in a viscosity-reducing amount; and wherein the mammalexperiences less pain with administration of the therapeutic formulationcomprising the excipient compound than that with administration of acontrol therapeutic formulation, wherein the control therapeuticformulation does not contain the excipient compound and is otherwiseidentical to the therapeutic formulation.

Disclosed herein, in embodiments, are methods of improving stability ofa liquid protein formulation, comprising: preparing a liquid proteinformulation comprising a therapeutic protein and an excipient compoundselected from the group selected from the group consisting of hinderedamines, anionic aromatics, functionalized amino acids, oligopeptides,and short-chain organic acids, and low molecular weight aliphaticpolyacids, wherein the liquid protein formulation demonstrates improvedstability compared to a control liquid protein formulation, wherein thecontrol liquid protein formulation does not contain the excipientcompound and is otherwise identical to the liquid protein formulation.The stability of the liquid formulation can be a cold storage conditionsstability, a room temperature stability or an elevated temperaturestability.

Also disclosed herein, in embodiments, are liquid formulationscomprising a protein and an excipient compound selected from the groupconsisting of hindered amines, anionic aromatics, functionalized aminoacids, oligopeptides, short-chain organic acids, and low molecularweight aliphatic polyacids, wherein the presence of the excipientcompound in the formulation results in improved protein-proteininteraction characteristics as measured by the protein diffusioninteraction parameter kD, or the second virial coefficient B22. Inembodiments, the formulation is a therapeutic formulation, and comprisesa therapeutic protein. In embodiments, the formulation is anon-therapeutic formulation, and comprises a non-therapeutic protein.

Further disclosed herein, in embodiments, are methods of improving aprotein-related process comprising providing the liquid formulationdescribed above, and employing it in a processing method. Inembodiments, the processing method includes filtration, pumping, mixing,centrifugation, membrane separation, lyophilization, or chromatography.In embodiments, the processing method is selected from the groupconsisting of cell culture harvest, chromatography, viral inactivation,and filtration. In embodiments, the processing method is achromatography process or a filtration process. In embodiments, thefiltration process is a virus filtration process or anultrafiltration/diafiltration process.

Also disclosed herein are methods of improving a parameter of aprotein-related process, comprising providing a viscosity-reducingexcipient additive comprising at least one excipient compound selectedfrom the group consisting of hindered amines, anionic aromatics,functionalized amino acids, oligopeptides, short-chain organic acids,low molecular weight aliphatic polyacids, and diones and sulfones, andadding a viscosity-reducing amount of the at least one excipientcompound to a carrier solution for the protein-related process, whereinthe carrier solution contains a protein of interest, thereby improvingthe parameter. In embodiments, the parameter can be selected from thegroup consisting of cost of protein production, amount of proteinproduction, rate of protein production, and efficiency of proteinproduction. The parameter can be a proxy parameter. In embodiments, theprotein-related process is an upstream processing process. The carriersolution for the upstream processing process can be a cell culturemedium. In embodiments, if the carrier solution is a cell culturemedium, the step of adding the excipient additive to the carriersolution comprises a first substep of adding the excipient additive to asupplemental medium to form an excipient-containing supplemental medium,and a second substep of adding the excipient-containing supplementalmedium to the cell culture medium. In other embodiments, theprotein-related process is a downstream processing process. Thedownstream process can be a chromatography process, and thechromatography process can be a Protein-A chromatography process. Inembodiments, the chromatography process recovers the protein ofinterest, wherein the protein of interest is characterized by animproved protein-related parameter selected from the group consisting ofimproved purity, improved yield, fewer particles, less misfolding, orless aggregation, as compared to a control solution. In embodiments, theimproved protein-related parameter is improved yield of the protein ofinterest from the chromatography process. In other embodiments, theprotein-related process is a process selected from the group consistingof filtration, injection, syringing, pumping, mixing, centrifugation,membrane separation, and lyophilization, and the selected process canrequire less force than a control process. In embodiments, theprotein-related process is selected from the group consisting of a cellculture process, a cell culture harvesting process, a chromatographyprocess, a viral inactivation process, and a filtration process. Inembodiments, the protein-related process is the viral inactivationprocess, and the viral inactivation process is conducted at a pH levelof about 2.5 to about 5.0, or the viral inactivation process isconducted at a higher pH than the control process. In other embodiments,the protein-related process is the filtration process. The filtrationprocess can be a virus removal filtration process or anultrafiltration/diafiltration process. The filtration process can becharacterized by an improved filtration-related parameter. The improvedfiltration-related parameter can be a faster filtration rate than thefiltration rate of the control solution. The improved filtration-relatedparameter can be a production of a smaller amount of aggregated proteinthan the amount of aggregated protein produced by a control filtrationprocess. The improved filtration-related parameter can be a higher masstransfer efficiency than the mass transfer efficiency of the controlfiltration process. The improved filtration-related parameter can be ahigher concentration or a higher yield of the target protein than aconcentration or yield of the target protein produced by the controlfiltration process.

Further disclosed herein are methods as described above, wherein theviscosity-reducing excipient additive comprises two or more excipientcompounds. In embodiments, the at least one excipient compound is ahindered amine. In embodiments, the at least one excipient compound isselected from the group consisting of caffeine, saccharin, acesulfamepotassium, aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone,2-pyrrolidinone, niacinamide, and imidazole. In embodiments, the atleast one excipient compound is selected from the group consisting ofcaffeine, taurine, niacinamide, and imidazole. In embodiments, the atleast one excipient compound is an anionic aromatic excipient, and, insome embodiments, the anionic aromatic excipient can be4-hydroxybenzenesulfonic acid. In embodiments, the viscosity-reducingamount is between about 1 mg/mL to about 100 mg/mL of the at least oneexcipient compound, or the viscosity-reducing amount is between about 1mM to about 400 mM of the at least one excipient compound, or theviscosity-reducing amount is an amount from about 2 mM to about 150 mM.In embodiments, the carrier solution comprises an additional agentselected from the group consisting of preservatives, sugars,polysaccharides, arginine, proline, surfactants, stabilizers, andbuffers. In embodiments, the protein of interest is a therapeuticprotein, and the therapeutic protein can be a recombinant protein, orcan be selected from the group consisting of a monoclonal antibody, apolyclonal antibody, an antibody fragment, a fusion protein, a PEGylatedprotein, an antibody-drug conjugate, a synthetic polypeptide, a proteinfragment, a lipoprotein, an enzyme, and a structural peptide. Inembodiments, the methods further comprise a step of adding a secondviscosity-reducing excipient to the carrier solution, wherein the stepof adding the second viscosity-reducing compound adds an additionalimprovement to the parameter.

In addition, carrier solutions are disclosed herein, comprising a liquidmedium in which is dissolved a protein of interest, and aviscosity-reducing additive, wherein the carrier solution has a lowerviscosity that that of a control solution. The carrier solution canfurther comprise an additional agent selected from the group consistingof preservatives, sugars, polysaccharides, arginine, proline,surfactants, stabilizers, and buffers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a block diagram showing the steps in a fermentationprocess (an “upstream processing”) for producing therapeutic proteins,for example monoclonal antibodies.

FIG. 2 presents a block diagram showing the steps in a purificationprocess (a “downstream processing”) for producing therapeutic proteins,for example monoclonal antibodies.

DETAILED DESCRIPTION

Disclosed herein are formulations and methods for their production thatpermit the delivery of concentrated protein solutions. In embodiments,the approaches disclosed herein can yield a lower viscosity liquidformulation or a higher concentration of therapeutic or nontherapeuticproteins in the liquid formulation, as compared to traditional proteinsolutions. In embodiments, the approaches disclosed herein can yield aliquid formulation having improved stability when compared to atraditional protein solution. A stable formulation is one in which theprotein contained therein substantially retains its physical andchemical stability and its therapeutic or nontherapeutic efficacy uponstorage under storage conditions, whether cold storage conditions, roomtemperature conditions, or elevated temperature storage conditions.Advantageously, a stable formulation can also offer protection againstaggregation or precipitation of the proteins dissolved therein. Forexample, the cold storage conditions can entail storage in arefrigerator or freezer. In some examples, cold storage conditions canentail storage at a temperature of 10° or less. In additional examples,the cold storage conditions entail storage at a temperature from about2° to about 10° C. In other examples, the cold storage conditions entailstorage at a temperature of about 4° C. In additional examples, the coldstorage conditions entail storage at freezing temperature such as about−20° C. or lower. In another example, cold storage conditions entailstorage at a temperature of about −20° C. to about 0° C. The roomtemperature storage conditions can entail storage at ambienttemperatures, for example, from about 10° C. to about 30° C. Elevatedstorage conditions can entail storage at a temperature greater. Elevatedtemperature stability, for example at temperatures from about 30° C. toabout 50° C., can be used as part an accelerated aging study to predictthe long-term storage at typical ambient (10-30° C.) conditions.

It is well known to those skilled in the art of polymer science andengineering that proteins in solution tend to form entanglements, whichcan limit the translational mobility of the entangled chains andinterfere with the protein's therapeutic or nontherapeutic efficacy. Inembodiments, excipient compounds as disclosed herein can suppressprotein clustering due to specific interactions between the excipientcompound and the target protein in solution. Excipient compounds asdisclosed herein can be natural or synthetic, and desirably aresubstances that the FDA generally recognizes as safe (“GRAS”).

1. Definitions

For the purpose of this disclosure, the term “protein” refers to asequence of amino acids having a chain length long enough to produce adiscrete tertiary structure, typically having a molecular weight between1-3000 kD. In some embodiments, the molecular weight of the protein isbetween about 50-200 kD; in other embodiments, the molecular weight ofthe protein is between about 20-1000 kD or between about 20-2000 kD. Incontrast to the term “protein,” the term “peptide” refers to a sequenceof amino acids that does not have a discrete tertiary structure. A widevariety of biopolymers are included within the scope of the term“protein.” For example, the term “protein” can refer to therapeutic ornon-therapeutic proteins, including antibodies, aptamers, fusionproteins, PEGylated proteins, synthetic polypeptides, protein fragments,lipoproteins, enzymes, structural peptides, and the like.

As non-limiting examples, therapeutic proteins can include mammalianproteins such as hormones and prohormones (e.g., insulin and proinsulin,glucagon, calcitonin, thyroid hormones (T3 or T4 or thyroid-stimulatinghormone), parathyroid hormone, follicle-stimulating hormone, luteinizinghormone, growth hormone, growth hormone releasing factor, and the like);clotting and anti-clotting factors (e.g., tissue factor, vonWillebrand's factor, Factor VIIIC, Factor IX, protein C, plasminogenactivators (urokinase, tissue-type plasminogen activators), thrombin);cytokines, chemokines, and inflammatory mediators; interferons;colony-stimulating factors; interleukins (e.g., IL-1 through IL-10);growth factors (e.g., vascular endothelial growth factors, fibroblastgrowth factor, platelet-derived growth factor, transforming growthfactor, neurotrophic growth factors, insulin-like growth factor, and thelike); albumins; collagens and elastins; hematopoietic factors (e.g.,erythropoietin, thrombopoietin, and the like); osteoinductive factors(e.g., bone morphogenetic protein); receptors (e.g., integrins,cadherins, and the like); surface membrane proteins; transport proteins;regulatory proteins; antigenic proteins (e.g., a viral component thatacts as an antigen); and antibodies. The term “antibody” is used hereinin its broadest sense, to include as non-limiting examples monoclonalantibodies (including, for example, full-length antibodies with animmunoglobulin Fc region), single-chain molecules, bi-specific andmulti-specific antibodies, diabodies, antibody-drug conjugates, antibodycompositions having polyepitopic specificity, polyclonal antibodies(such as polyclonal immunoglobulins used as therapies forimmune-compromised patients), and fragments of antibodies (including,for example, Fc, Fab, Fv, and F(ab′)2). Antibodies can also be termed“immunoglobulins.” An antibody is understood to be directed against aspecific protein or non-protein “antigen,” which is a biologicallyimportant material; the administration of a therapeutically effectiveamount of an antibody to a patient can complex with the antigen, therebyaltering its biological properties so that the patient experiences atherapeutic effect.

In embodiments, the proteins are PEGylated, meaning that they comprisepoly(ethylene glycol) (“PEG”) and/or poly(propylene glycol) (“PPG”)units. PEGylated proteins, or PEG-protein conjugates, have found utilityin therapeutic applications due to their beneficial properties such assolubility, pharmacokinetics, pharmacodynamics, immunogenicity, renalclearance, and stability. Non-limiting examples of PEGylated proteinsare PEGylated interferons (PEG-IFN), PEGylated anti-VEGF, PEG proteinconjugate drugs, Adagen, Pegaspargase, Pegfilgrastim, Pegloticase,Pegvisomant, PEGylated epoetin-β, and Certolizumab pegol.

PEGylated proteins can be synthesized by a variety of methods such as areaction of protein with a PEG reagent having one or more reactivefunctional groups. The reactive functional groups on the PEG reagent canform a linkage with the protein at targeted protein sites such aslysine, histidine, cysteine, and the N-terminus. Typical PEGylationreagents have reactive functional groups such as aldehyde, maleimide, orsuccinimide groups that have specific reactivity with targeted aminoacid residues on proteins. The PEGylation reagents can have a PEG chainlength from about 1 to about 1000 PEG and/or PPG repeating units. Othermethods of PEGylation include glyco-PEGylation, where the protein isfirst glycosylated and then the glycosylated residues are PEGylated in asecond step. Certain PEGylation processes are assisted by enzymes likesialyltransferase and transglutaminase.

While the PEGylated proteins can offer therapeutic advantages overnative, non-PEGylated proteins, these materials can have physical orchemical properties that make them difficult to purify, dissolve,filter, concentrate, and administer. The PEGylation of a protein canlead to a higher solution viscosity compared to the native protein, andthis generally requires the formulation of PEGylated protein solutionsat lower concentrations.

It is desirable to formulate protein therapeutics in stable, lowviscosity solutions so they can be administered to patients in a minimalinjection volume. For example, the subcutaneous (SC) or intramuscular(IM) injection of drugs generally requires a small injection volume,preferably less than 2 mL. The SC and IM injection routes are wellsuited to self-administered care, and this is a less costly and moreaccessible form of treatment compared with intravenous (IV) injectionwhich is only conducted under direct medical supervision. Formulationsfor SC or IM injection require a low solution viscosity, generally below30 cP, and preferably below 20 cP, to allow easy flow of the therapeuticsolution through a narrow-gauge needle. This combination of smallinjection volume and low viscosity requirements present a challenge tothe use of PEGylated protein therapeutics in SC or IM injection routes.

Those proteins having therapeutic effects may be termed “therapeuticproteins”; formulations containing therapeutic proteins intherapeutically effective amounts may be termed “therapeuticformulations.” The therapeutic protein contained in a therapeuticformulation may also be termed its “protein active ingredient.”Typically, a therapeutic formulation comprises a therapeuticallyeffective amount of a protein active ingredient and an excipient, withor without other optional components. As used herein, the term“therapeutic” includes both treatments of existing disorders andpreventions of disorders. Therapeutic proteins include, for example,proteins such as bevacizumab, trastuzumab, adalimumab, infliximab,etanercept, darbepoetin alfa, epoetin alfa, cetuximab, filgrastim, andrituximab. Other therapeutic proteins will be familiar to those havingordinary skill in the art.

A “treatment” includes any measure intended to cure, heal, alleviate,improve, remedy, or otherwise beneficially affect the disorder,including preventing or delaying the onset of symptoms and/oralleviating or ameliorating symptoms of the disorder. Those patients inneed of a treatment include both those who already have a specificdisorder, and those for whom the prevention of a disorder is desirable.A disorder is any condition that alters the homeostatic wellbeing of amammal, including acute or chronic diseases, or pathological conditionsthat predispose the mammal to an acute or chronic disease. Non-limitingexamples of disorders include cancers, metabolic disorders (e.g.,diabetes), allergic disorders (e.g., asthma), dermatological disorders,cardiovascular disorders, respiratory disorders, hematologicaldisorders, musculoskeletal disorders, inflammatory or rheumatologicaldisorders, autoimmune disorders, gastrointestinal disorders, urologicaldisorders, sexual and reproductive disorders, neurological disorders,and the like. The term “mammal” for the purposes of treatment can referto any animal classified as a mammal, including humans, domesticanimals, pet animals, farm animals, sporting animals, working animals,and the like. A “treatment” can therefore include both veterinary andhuman treatments. For convenience, the mammal undergoing such“treatment” can be referred to as a “patient.” In certain embodiments,the patient can be of any age, including fetal animals in utero.

In embodiments, a treatment involves providing a therapeuticallyeffective amount of a therapeutic formulation to a mammal in needthereof. A “therapeutically effective amount” is at least the minimumconcentration of the therapeutic protein administered to the mammal inneed thereof, to effect a treatment of an existing disorder or aprevention of an anticipated disorder (either such treatment or suchprevention being a “therapeutic intervention”). Therapeuticallyeffective amounts of various therapeutic proteins that may be includedas active ingredients in the therapeutic formulation may be familiar inthe art; or, for therapeutic proteins discovered or applied totherapeutic interventions hereinafter, the therapeutically effectiveamount can be determined by standard techniques carried out by thosehaving ordinary skill in the art, using no more than routineexperimentation.

Those proteins used for non-therapeutic purposes (i.e., purposes notinvolving treatments), such as household, nutrition, commercial, andindustrial applications, may be termed “non-therapeutic proteins.”Formulations containing non-therapeutic proteins may be termed“non-therapeutic formulations”. The non-therapeutic proteins can bederived from plant sources, animal sources, or produced from cellcultures; they also can be enzymes or structural proteins. Thenon-therapeutic proteins can be used in in household, nutrition,commercial, and industrial applications such as catalysts, human andanimal nutrition, processing aids, cleaners, and waste treatment.

An important category of non-therapeutic biopolymers is enzymes. Enzymeshave a number of non-therapeutic applications, for example, ascatalysts, human and animal nutritional ingredients, processing aids,cleaners, and waste treatment agents. Enzyme catalysts are used toaccelerate a variety of chemical reactions. Examples of enzyme catalystsfor non-therapeutic uses include catalases, oxidoreductases,transferases, hydrolases, lyases, isomerases, and ligases. Human andanimal nutritional uses of enzymes include nutraceuticals, nutritivesources of protein, chelation or controlled delivery of micronutrients,digestion aids, and supplements; these can be derived from amylase,protease, trypsin, lactase, and the like. Enzymatic processing aids areused to improve the production of food and beverage products inoperations like baking, brewing, fermenting, juice processing, andwinemaking. Examples of these food and beverage processing aids includeamylases, cellulases, pectinases, glucanases, lipases, and lactases.Enzymes can also be used in the production of biofuels. Ethanol forbiofuels, for example, can be aided by the enzymatic degradation ofbiomass feedstocks such as cellulosic and lignocellulosic materials. Thetreatment of such feedstock materials with cellulases and ligninasestransforms the biomass into a substrate that can be fermented intofuels. In other commercial applications, enzymes are used as detergents,cleaners, and stain lifting aids for laundry, dish washing, surfacecleaning, and equipment cleaning applications. Typical enzymes for thispurpose include proteases, cellulases, amylases, and lipases. Inaddition, non-therapeutic enzymes are used in a variety of commercialand industrial processes such as textile softening with cellulases,leather processing, waste treatment, contaminated sediment treatment,water treatment, pulp bleaching, and pulp softening and debonding.Typical enzymes for these purposes are amylases, xylanases, cellulases,and ligninases.

Other examples of non-therapeutic biopolymers include fibrous orstructural proteins such as keratins, collagen, gelatin, elastin,fibroin, actin, tubulin, or the hydrolyzed, degraded, or derivatizedforms thereof. These materials are used in the preparation andformulation of food ingredients such as gelatin, ice cream, yogurt, andconfections; they area also added to foods as thickeners, rheologymodifiers, mouthfeel improvers, and as a source of nutritional protein.In the cosmetics and personal care industry, collagen, elastin, keratin,and hydrolyzed keratin are widely used as ingredients in skin care andhair care formulations. Still other examples of non-therapeuticbiopolymers are whey proteins such as beta-lactoglobulin,alpha-lactalbumin, and serum albumin. These whey proteins are producedin mass scale as a byproduct from dairy operations and have been usedfor a variety of non-therapeutic applications.

2. Measurements

In embodiments, the protein-containing formulations described herein areresistant to monomer loss as measured by size exclusion chromatography(SEC) analysis. In SEC analysis as used herein, the main analyte peak isgenerally associated with the target protein contained in theformulation, and this main peak of the protein is referred to as themonomer peak. The monomer peak represents the amount of target protein,e.g., a protein active ingredient, in the monomeric state, as opposed toaggregated (dimeric, trimeric, oligomeric, etc.) or fragmented states.The monomer peak area can be compared with the total area of themonomer, aggregate, and fragment peaks associated with the targetprotein. Thus, the stability of a protein-containing formulation can beobserved by the relative amount of monomer after an elapsed time; animprovement in stability of a protein-containing formulation of theinvention can therefore be measured as a higher percent monomer after acertain elapsed time, as compared to the percent monomer in a controlformulation that does not contain the excipient.

In embodiments, an ideal stability result is to have from 98 to 100%monomer peak as determined by SEC analysis. In embodiments, animprovement in stability of a protein-containing formulation of theinvention can be measured as a higher percent monomer after exposure toa stress condition, as compared to the percent monomer in a controlformulation that does not contain the excipient when such controlformulation is exposed to the same stress condition. In embodiments, thestress conditions can be a low temperature storage, high temperaturestorage, exposure to air, exposure to gas bubbles, exposure to shearconditions, or exposure to freeze/thaw cycles.

In embodiments, the protein-containing formulations as described hereinare resistant to an increase in protein particle size as measured bydynamic light scattering (DLS) analysis. In DLS analysis as used herein,the particle size of the protein in the protein-containing formulationcan be observed as a median particle diameter. Ideally, the medianparticle diameter of the target protein should be relatively unchangedwhen subjected to DLS analysis, since the particle diameter representsthe active component in the monomeric state, as opposed to aggregated(dimeric, trimeric, oligomeric, etc.) or fragmented states. An increaseof the median particle diameter could represent an aggregated protein.Thus, the stability of a protein-containing formulation can be observedby the relative change in median particle diameter after an elapsedtime.

In embodiments, the protein-containing formulations as described hereinare resistant to forming a polydisperse particle size distribution asmeasured by dynamic light scattering (DLS) analysis. In embodiments, aprotein-containing formulation can contain a monodisperse particle sizedistribution of colloidal protein particles. In embodiments, an idealstability result is to have less than a 10% change in the medianparticle diameter compared to the initial median particle diameter ofthe formulation. In embodiments, an improvement in stability of aprotein-containing formulation of the invention can be measured as alower percent change of the median particle diameter after a certainelapsed time, as compared to the median particle diameter in a controlformulation that does not contain the excipient. In embodiments, animprovement in stability of a protein-containing formulation of theinvention can be measured as a lower percent change of the medianparticle diameter after exposure to a stress condition, as compared tothe percent change of the median particle diameter in a controlformulation that does not contain the excipient when such controlformulation is exposed to the same stress condition. In embodiments, thestress conditions can be a low temperature storage, high temperaturestorage, exposure to air, exposure to gas bubbles, exposure to shearconditions, or exposure to freeze/thaw cycles. In embodiments, animprovement in stability of a protein-containing formulation therapeuticformulation of the invention can be measured as a less polydisperseparticle size distribution as measured by DLS, as compared to thepolydispersity of the particle size distribution in a controlformulation that does not contain the excipient when such controlformulation is exposed to the same stress condition.

In embodiments, the protein-containing formulations of the invention areresistant to precipitation as measured by turbidity, light scattering,and/or particle counting analysis. In turbidity, light scattering, orparticle counting analysis, a lower value generally represents a lowernumber of suspended particles in a formulation. An increase ofturbidity, light scattering, or particle counting can indicate that thesolution of the target protein is not stable. Thus, the stability of aprotein-containing formulation can be observed by the relative amount ofturbidity, light scattering, or particle counting after an elapsed time.In embodiments, an ideal stability result is to have a low andrelatively constant turbidity, light scattering, or particle countingvalue. In embodiments, an improvement in stability of aprotein-containing formulation of the invention can be measured as alower turbidity, lower light scattering, or lower particle count after acertain elapsed time, as compared to the turbidity, light scattering, orparticle count values in a control formulation that does not contain theexcipient. In embodiments, an improvement in stability of aprotein-containing formulation as described herein can be measured as alower turbidity, lower light scattering, or lower particle count afterexposure to a stress condition, as compared to the turbidity, lightscattering, or particle count in a control formulation that does notcontain the excipient when such control formulation is exposed to thesame stress condition. In embodiments, the stress conditions can be alow temperature storage, high temperature storage, exposure to air,exposure to gas bubbles, exposure to shear conditions, or exposure tofreeze/thaw cycles.

3. Therapeutic Formulations

In one aspect, the formulations and methods disclosed herein providestable liquid formulations of improved or reduced viscosity, comprisinga therapeutic protein in a therapeutically effective amount and anexcipient compound. In embodiments, the formulation can improve thestability while providing an acceptable concentration of activeingredients and an acceptable viscosity. In embodiments, the formulationprovides an improvement in stability when compared to a controlformulation; for the purposes of this disclosure, a control formulationis a formulation containing the protein active ingredient that isidentical on a dry weight basis in every way to the therapeuticformulation except that it lacks the excipient compound. In embodiments,improved stability of the protein containing formulation is in the formof lower percentage of soluble aggregates, particulates, subvisibleparticles, or gel formation, compared to a control formulation.

It is understood that the viscosity of a liquid protein formulation canbe affected by a variety of factors, including but not limited to: thenature of the protein itself (e.g., enzyme, antibody, receptor, fusionprotein, etc.); its size, three-dimensional structure, chemicalcomposition, and molecular weight; its concentration in the formulation;the components of the formulation besides the protein; the desired pHrange; the storage conditions for the formulation; and the method ofadministering the formulation to the patient. Therapeutic proteins mostsuitable for use with the excipient compounds described herein arepreferably essentially pure, i.e., free from contaminating proteins. Inembodiments, an “essentially pure” therapeutic protein is a proteincomposition comprising at least 90% by weight of the therapeuticprotein, or preferably at least 95% by weight, or more preferably, atleast 99% by weight, all based on the total weight of therapeuticproteins and contaminating proteins in the composition. For the purposesof clarity, a protein added as an excipient is not intended to beincluded in this definition. The therapeutic formulations describedherein are intended for use as pharmaceutical-grade formulations, i.e.,formulations intended for use in treating a mammal, in such a form thatthe desired therapeutic efficacy of the protein active ingredient can beachieved, and without containing components that are toxic to the mammalto whom the formulation is to be administered.

In embodiments, the therapeutic formulation contains at least 25 mg/mLof protein active ingredient. In other embodiments, the therapeuticformulation contains at least 100 mg/mL of protein active ingredient. Inother embodiments, the therapeutic formulation contains at least 200mg/mL of protein active ingredient. In yet other embodiments, thetherapeutic formulation solution contains at least 300 mg/mL of proteinactive ingredient. Generally, the excipient compounds disclosed hereinare added to the therapeutic formulation in an amount between about 5 toabout 300 mg/mL. In embodiments, the excipient compound can be added inan amount of about 10 to about 200 mg/mL. In embodiments, the excipientcompound can be added in an amount of about 20 to about 100 mg/mL. Inembodiments, the excipient can be added in an amount of about 25 toabout 75 mg/mL.

Excipient compounds of various molecular weights are selected forspecific advantageous properties when combined with the protein activeingredient in a formulation. Examples of therapeutic formulationscomprising excipient compounds are provided below. In embodiments, theexcipient compound has a molecular weight of <5000 Da. In embodiments,the excipient compound has a molecular weight of <1000 Da. Inembodiments, the excipient compound has a molecular weight of <500 Da.

In embodiments, the excipient compounds disclosed herein is added to thetherapeutic formulation in a viscosity-reducing amount. In embodiments,a viscosity-reducing amount is the amount of an excipient compound thatreduces the viscosity of the formulation at least 10% when compared to acontrol formulation; for the purposes of this disclosure, a controlformulation is a formulation containing the protein active ingredientthat is identical on a dry weight basis in every way to the therapeuticformulation except that it lacks the excipient compound. In embodiments,the viscosity-reducing amount is the amount of an excipient compoundthat reduces the viscosity of the formulation at least 30% when comparedto the control formulation. In embodiments, the viscosity-reducingamount is the amount of an excipient compound that reduces the viscosityof the formulation at least 50% when compared to the controlformulation. In embodiments, the viscosity-reducing amount is the amountof an excipient compound that reduces the viscosity of the formulationat least 70% when compared to the control formulation. In embodiments,the viscosity-reducing amount is the amount of an excipient compoundthat reduces the viscosity of the formulation at least 90% when comparedto the control formulation.

In embodiments, the viscosity-reducing amount yields a therapeuticformulation having a viscosity of less than 100 cP. In otherembodiments, the therapeutic formulation has a viscosity of less than 50cP. In other embodiments, the therapeutic formulation has a viscosity ofless than 20 cP. In yet other embodiments, the therapeutic formulationhas a viscosity of less than 10 cP. The term “viscosity” as used hereinrefers to a dynamic viscosity value when measured by the methodsdisclosed herein.

Therapeutic formulations in accordance with this disclosure have certainadvantageous properties. In embodiments, the therapeutic formulationsare resistant to shear degradation, phase separation, clouding out,oxidation, deamidation, aggregation, precipitation, and denaturing. Inembodiments, the therapeutic formulations are processed, purified,stored, syringed, dosed, filtered, and centrifuged more effectively,compared with a control formulation. In embodiments, the therapeuticformulations are administered to a patient at high concentration oftherapeutic protein. In embodiments, the therapeutic formulations areadministered to patients with less discomfort than would be experiencedwith a similar formulation lacking the therapeutic excipient. Inembodiments, the therapeutic formulations are administered as a depotinjection. In embodiments, the therapeutic formulations extend thehalf-life of the therapeutic protein in the body.

These features of therapeutic formulations as disclosed herein wouldpermit the administration of such formulations by intramuscular orsubcutaneous injection in a clinical situation, i.e., a situation wherepatient acceptance of an intramuscular injection would include the useof small-bore needles typical for IM/SC purposes and the use of atolerable (for example, 2-3 cc) injected volume, and where theseconditions result in the administration of an effective amount of theformulation in a single injection at a single injection site. Bycontrast, injection of a comparable dosage amount of the therapeuticprotein using conventional formulation techniques would be limited bythe higher viscosity of the conventional formulation, so that a SC/IMinjection of the conventional formulation would not be suitable for aclinical situation. High concentration solutions of therapeutic proteinsformulated with the excipient compounds described herein can beadministered to patients using syringes or pre-filled syringes.

In embodiments, the therapeutic excipient has antioxidant propertiesthat stabilize the therapeutic protein against oxidative damage. Inembodiments, the therapeutic formulation is stored at ambienttemperatures, or for extended time at refrigerator conditions withoutappreciable loss of potency for the therapeutic protein. In embodiments,the therapeutic formulation is dried down for storage until it isneeded; then it is reconstituted with an appropriate solvent, e.g.,water. Advantageously, the formulations prepared as described herein canbe stable over a prolonged period of time, from months to years. Whenexceptionally long periods of storage are desired, the formulations canbe preserved in a freezer (and later reactivated) without fear ofprotein denaturation. In embodiments, formulations can be prepared forlong-term storage that do not require refrigeration.

Methods for preparing therapeutic formulations may be familiar toskilled artisans. The therapeutic formulations of the present inventioncan be prepared, for example, by adding the excipient compound to theformulation before or after the therapeutic protein is added to thesolution. The therapeutic formulation can, for example, be produced bycombining the therapeutic protein and the excipient at a first (lower)concentration and then processed by filtration or centrifugation toproduce a second (higher) concentration of the therapeutic protein.Therapeutic formulations can be made with one or more of the excipientcompounds with chaotropes, kosmotropes, hydrotropes, and salts.Therapeutic formulations can be made with one or more of the excipientcompounds using techniques such as encapsulation, dispersion, liposome,vesicle formation, and the like. Methods for preparing therapeuticformulations comprising the excipient compounds disclosed herein caninclude combinations of the excipient compounds. In embodiments,combinations of excipients can produce benefits in lower viscosity,improved stability, or reduced injection site pain. Other additives maybe introduced into the therapeutic formulations during theirmanufacture, including preservatives, surfactants, sugars, sucrose,trehalose, polysaccharides, arginine, proline, hyaluronidase,stabilizers, buffers, and the like. As used herein, a pharmaceuticallyacceptable excipient compound is one that is non-toxic and suitable foranimal and/or human administration.

4. Non-Therapeutic Formulations

In one aspect, the formulations and methods disclosed herein providestable liquid formulations of improved or reduced viscosity, comprisinga non-therapeutic protein in an effective amount and an excipientcompound. In embodiments, the formulation improves the stability whileproviding an acceptable concentration of active ingredients and anacceptable viscosity. In embodiments, the formulation provides animprovement in stability when compared to a control formulation; for thepurposes of this disclosure, a control formulation is a formulationcontaining the protein active ingredient that is identical on a dryweight basis in every way to the non-therapeutic formulation except thatit lacks the excipient compound.

It is understood that the viscosity of a liquid protein formulation canbe affected by a variety of factors, including but not limited to: thenature of the protein itself (e.g., enzyme, structural protein, degreeof hydrolysis, etc.); its size, three-dimensional structure, chemicalcomposition, and molecular weight; its concentration in the formulation;the components of the formulation besides the protein; the desired pHrange; and the storage conditions for the formulation.

In embodiments, the non-therapeutic formulation contains at least 25mg/mL of protein active ingredient. In other embodiments, thenon-therapeutic formulation contains at least 100 mg/mL of proteinactive ingredient. In other embodiments, the non-therapeutic formulationcontains at least 200 mg/mL of protein active ingredient. In yet otherembodiments, the non-therapeutic formulation solution contains at least300 mg/mL of protein active ingredient. Generally, the excipientcompounds disclosed herein are added to the non-therapeutic formulationin an amount between about 5 to about 300 mg/mL. In embodiments, theexcipient compound is added in an amount of about 10 to about 200 mg/mL.In embodiments, the excipient compound is added in an amount of about 20to about 100 mg/mL. In embodiments, the excipient is added in an amountof about 25 to about 75 mg/mL.

Excipient compounds of various molecular weights are selected forspecific advantageous properties when combined with the protein activeingredient in a formulation. Examples of non-therapeutic formulationscomprising excipient compounds are provided below. In embodiments, theexcipient compound has a molecular weight of <5000 Da. In embodiments,the excipient compound has a molecular weight of <1000 Da. Inembodiments, the excipient compound has a molecular weight of <500 Da.

In embodiments, the excipient compounds disclosed herein is added to thenon-therapeutic formulation in a viscosity-reducing amount. Inembodiments, a viscosity-reducing amount is the amount of an excipientcompound that reduces the viscosity of the formulation at least 10% whencompared to a control formulation; for the purposes of this disclosure,a control formulation is a formulation containing the protein activeingredient that is identical on a dry weight basis in every way to thetherapeutic formulation except that it lacks the excipient compound. Inembodiments, the viscosity-reducing amount is the amount of an excipientcompound that reduces the viscosity of the formulation at least 30% whencompared to the control formulation. In embodiments, theviscosity-reducing amount is the amount of an excipient compound thatreduces the viscosity of the formulation at least 50% when compared tothe control formulation. In embodiments, the viscosity-reducing amountis the amount of an excipient compound that reduces the viscosity of theformulation at least 70% when compared to the control formulation. Inembodiments, the viscosity-reducing amount is the amount of an excipientcompound that reduces the viscosity of the formulation at least 90% whencompared to the control formulation.

In embodiments, the viscosity-reducing amount yields a non-therapeuticformulation having a viscosity of less than 100 cP. In otherembodiments, the non-therapeutic formulation has a viscosity of lessthan 50 cP. In other embodiments, the non-therapeutic formulation has aviscosity of less than 20 cP. In yet other embodiments, thenon-therapeutic formulation has a viscosity of less than 10 cP. The term“viscosity” as used herein refers to a dynamic viscosity value.

Non-therapeutic formulations in accordance with this disclosure can havecertain advantageous properties. In embodiments, the non-therapeuticformulations are resistant to shear degradation, phase separation,clouding out, oxidation, deamidation, aggregation, precipitation, anddenaturing. In embodiments, the therapeutic formulations can beprocessed, purified, stored, pumped, filtered, and centrifuged moreeffectively, compared with a control formulation.

In embodiments, the non-therapeutic excipient has antioxidant propertiesthat stabilize the non-therapeutic protein against oxidative damage. Inembodiments, the non-therapeutic formulation is stored at ambienttemperatures, or for extended time at refrigerator conditions withoutappreciable loss of potency for the non-therapeutic protein. Inembodiments, the non-therapeutic formulation is dried down for storageuntil it is needed; then it can be reconstituted with an appropriatesolvent, e.g., water. Advantageously, the formulations prepared asdescribed herein is stable over a prolonged period of time, from monthsto years. When exceptionally long periods of storage are desired, theformulations are preserved in a freezer (and later reactivated) withoutfear of protein denaturation. In embodiments, formulations are preparedfor long-term storage that do not require refrigeration.

Methods for preparing non-therapeutic formulations comprising theexcipient compounds disclosed herein may be familiar to skilledartisans. For example, the excipient compound can be added to theformulation before or after the non-therapeutic protein is added to thesolution. The non-therapeutic formulation can be produced at a first(lower) concentration and then processed by filtration or centrifugationto produce a second (higher) concentration. Non-therapeutic formulationscan be made with one or more of the excipient compounds with chaotropes,kosmotropes, hydrotropes, and salts. Non-therapeutic formulations can bemade with one or more of the excipient compounds using techniques suchas encapsulation, dispersion, liposome, vesicle formation, and the like.Other additives can be introduced into the non-therapeutic formulationsduring their manufacture, including preservatives, surfactants,stabilizers, and the like.

5. Excipient Compounds

Several excipient compounds are described herein, each suitable for usewith one or more therapeutic or non-therapeutic proteins, and eachallowing the formulation to be composed so that it contains theprotein(s) at a high concentration. Some of the categories of excipientcompounds described below are: (1) hindered amines; (2) anionicaromatics; (3) functionalized amino acids; (4) oligopeptides; (5)short-chain organic acids; (6) low-molecular-weight aliphatic polyacids;and (7) diones and sulfones. Without being bound by theory, theexcipient compounds described herein are thought to associate withcertain fragments, sequences, structures, or sections of a therapeuticprotein that otherwise would be involved in inter-particle (i.e.,protein-protein) interactions. The association of these excipientcompounds with the therapeutic or non-therapeutic protein can mask theinter-protein interactions such that the proteins can be formulated inhigh concentration without causing excessive solution viscosity.Excipient compounds advantageously can be water-soluble, thereforesuitable for use with aqueous vehicles. In embodiments, the excipientcompounds have a water solubility of >10 mg/mL. In embodiments, theexcipient compounds have a water solubility of >100 mg/mL. Inembodiments, the excipient compounds have a water solubility of >500mg/mL. Advantageously for therapeutic proteins, the excipient compoundscan be derived from materials that are biologically acceptable and arenon-immunogenic, and are thus suitable for pharmaceutical use. Intherapeutic embodiments, the excipient compounds can be metabolized inthe body to yield biologically compatible and non-immunogenicbyproducts.

a. Excipient Compound Category 1: Hindered Amines

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with hindered amine small molecules as excipientcompounds. As used herein, the term “hindered amine” refers to a smallmolecule containing at least one bulky or sterically hindered group,consistent with the examples below. Hindered amines can be used in thefree base form, in the protonated form, or a combination of the two. Inprotonated forms, the hindered amines can be associated with an anioniccounterion such as chloride, hydroxide, bromide, iodide, fluoride,acetate, formate, phosphate, sulfate, or carboxylate. Hindered aminecompounds useful as excipient compounds can contain secondary amine,tertiary amine, quaternary ammonium, pyridinium, pyrrolidone,pyrrolidine, piperidine, morpholine, or guanidinium groups, such thatthe excipient compound has a cationic charge in aqueous solution atneutral pH. The hindered amine compounds also contain at least one bulkyor sterically hindered group, such as cyclic aromatic, cycloaliphatic,cyclohexyl, or alkyl groups. In embodiments, the sterically hinderedgroup can itself be an amine group such as a dialkylamine,trialkylamine, guanidinium, pyridinium, or quaternary ammonium group.Without being bound by theory, the hindered amine compounds are thoughtto associate with aromatic sections of the proteins such asphenylalanine, tryptophan, and tyrosine, by a cation pi interaction. Inembodiments, the cationic group of the hindered amine can have anaffinity for the electron rich pi structure of the aromatic amino acidresidues in the protein, so that they can shield these sections of theprotein, thereby decreasing the tendency of such shielded proteins toassociate and agglomerate.

In embodiments, the hindered amine excipient compounds has a chemicalstructure comprising imidazole, imidazoline, or imidazolidine groups, orsalts thereof, such as imidazole, 1-methylimidazole, 4-methylimidazole,1-hexyl-3-methylimidazolium chloride, histamine, 4-methylhistamine,alpha-methylhistamine, betahistine, beta-alanine,2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid,potassium urate, betazole, carnosine, aspartame, saccharin, acesulfamepotassium, xanthine, theophylline, theobromine, caffeine, and anserine.In embodiments, the hindered amine excipient compounds is selected fromthe group consisting of dimethylethanolamine, dimethylaminopropylamine,triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine,diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylenebiguanide, poly(hexamethylene biguanide), imidazole, dimethylglycine,agmatine, diazabicyclo[2.2.2]octane, tetramethylethylenediamine,N,N-dimethylethanolamine, ethanolamine phosphate, glucosamine, cholinechloride, phosphocholine, niacinamide, isonicotinamide, N,N-diethylnicotinamide, nicotinic acid sodium salt, tyramine, 3-aminopyridine,2,4,6-trimethylpyridine, 3-pyridine methanol, nicotinamide adenosinedinucleotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidinone,procaine, lidocaine, dicyandiamide-taurine adduct, 2-pyridylethylamine,dicyandiamide-benzyl amine adduct, dicyandiamide-alkylamine adduct,dicyandiamide-cycloalkylamine adduct, anddicyandiamide-aminomethanephosphonic acid adducts. In embodiments, ahindered amine compound consistent with this disclosure is formulated asa protonated ammonium salt. In embodiments, a hindered amine compoundconsistent with this disclosure is formulated as a salt with aninorganic anion or organic anion as the counterion. In embodiments, highconcentration solutions of therapeutic or non-therapeutic proteins areformulated with a combination of caffeine with a benzoic acid, ahydroxybenzoic acid, or a benzenesulfonic acid as excipient compounds.In embodiments, the hindered amine excipient compounds are metabolizedin the body to yield biologically compatible byproducts. In someembodiments, the hindered amine excipient compound is present in theformulation at a concentration of about 250 mg/ml or less. In additionalembodiments, the hindered amine excipient compound is present in theformulation at a concentration of about 10 mg/ml to about 200 mg/ml. Inyet additional aspects, the hindered amine excipient compound is presentin the formulation at a concentration of about 20 to about 120 mg/ml.

In embodiments, certain hindered amine excipient compounds can possessother pharmacological properties. As examples, xanthines are a categoryof hindered amines having independent pharmacological properties,including stimulant properties and bronchodilator properties whensystemically absorbed. Representative xanthines include caffeine,aminophylline, 3-isobutyl-1-methylxanthine, paraxanthine,pentoxifylline, theobromine, theophylline, and the like. Methylatedxanthines are understood to affect force of cardiac contraction, heartrate, and bronchodilation. In some embodiments, the xanthine excipientcompound is present in the formulation at a concentration of about 30mg/ml or less.

Another category of hindered amines having independent pharmacologicalproperties are the local injectable anesthetic compounds. Localinjectable anesthetic compounds are hindered amines that have athree-component molecular structure of (a) a lipophilic aromatic ring,(b) an intermediate ester or amide linkage, and (c) a secondary ortertiary amine. This category of hindered amines is understood tointerrupt neural conduction by inhibiting the influx of sodium ions,thereby inducing local anesthesia. The lipophilic aromatic ring for alocal anesthetic compound may be formed of carbon atoms (e.g., a benzenering) or it may comprise heteroatoms (e.g., a thiophene ring).Representative local injectable anesthetic compounds include, but arenot limited to, amylocaine, articaine, bupivicaine, butacaine,butanilicaine, chlorprocaine, cocaine, cyclomethycaine, dimethocaine,editocaine, hexylcaine, isobucaine, levobupivacaine, lidocaine,metabutethamine, metabutoxycaine, mepivacaine, meprylcaine,propoxycaine, prilocaine, procaine, piperocaine, tetracaine, trimecaine,and the like. The local injectable anesthetic compounds can havemultiple benefits in protein therapeutic formulations, such as reducedviscosity, improved stability, and reduced pain upon injection. In someembodiments, the local anesthetic compound is present in the formulationin a concentration of about 50 mg/ml or less.

In embodiments, a hindered amine having independent pharmacologicalproperties is used as an excipient compound in accordance with theformulations and methods described herein. In some embodiments, theexcipient compounds possessing independent pharmacological propertiesare present in an amount that does not have a pharmacological effectand/or that is not therapeutically effective. In other embodiments, theexcipient compounds possessing independent pharmacological propertiesare present in an amount that does have a pharmacological effect and/orthat is therapeutically effective. In certain embodiments, a hinderedamine having independent pharmacological properties is used incombination with another excipient compound that has been selected todecrease formulation viscosity, where the hindered amine havingindependent pharmacological properties is used to impart the benefits ofits pharmacological activity. For example, a local injectable anestheticcompound can be used to decrease formulation viscosity and also toreduce pain upon injection of the formulation. The reduction ofinjection pain can be caused by anesthetic properties; also, a lowerinjection force can be required when the viscosity is reduced by theexcipients. Alternatively, a local injectable anesthetic compound can beused to impart the desirable pharmacological benefit of decreased localsensation during formulation injection, while being combined withanother excipient compound that reduces the viscosity of theformulation.

b. Excipient Compound Category 2: Anionic Aromatics

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with anionic aromatic small molecule compounds asexcipient compounds. The anionic aromatic excipient compounds cancontain an aromatic functional group such as phenyl, benzyl, aryl,alkylbenzyl, hydroxybenzyl, phenolic, hydroxyaryl, heteroaromatic group,or a fused aromatic group. The anionic aromatic excipient compounds alsocan contain an anionic functional group such as carboxylate, oxide,phenoxide, sulfonate, sulfate, phosphonate, phosphate, or sulfide. Whilethe anionic aromatic excipients might be described as an acid, a sodiumsalt, or other, it is understood that the excipient can be used in avariety of salt forms. Without being bound by theory, an anionicaromatic excipient compound is thought to be a bulky, stericallyhindered molecule that can associate with cationic segments of aprotein, so that they can shield these sections of the protein, therebydecreasing the interactions between protein molecules that render theprotein-containing formulation viscous.

In embodiments, examples of anionic aromatic excipient compounds includecompounds such as salicylic acid, aminosalicylic acid, hydroxybenzoicacid, aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid,hydroxybenzenesulfonic acid, naphthalenesulfonic acid,naphthalenedisulfonic acid, hydroquinone sulfonic acid, sulfanilic acid,vanillic acid, vanillin, vanillin-taurine adduct, aminophenol,anthranilic acid, cinnamic acid, coumaric acid, adenosine monophosphate,indole acetic acid, potassium urate, furan dicarboxylic acid,furan-2-acrylic acid, 2-furanpropionic acid, sodium phenylpyruvate,sodium hydroxyphenylpyruvate, dihydroxybenzoic acid, trihydroxybenzoicacid, pyrogallol, benzoic acid, and the salts of the foregoing acids. Inembodiments, the anionic aromatic excipient compounds are formulated inthe ionized salt form. In embodiments, an anionic aromatic compound isformulated as the salt of a hindered amine, such asdimethylcyclohexylammonium hydroxybenzoate. In embodiments, the anionicaromatic excipient compounds are formulated with various counterionssuch as organic cations. In embodiments, high concentration solutions oftherapeutic or non-therapeutic proteins is formulated with anionicaromatic excipient compounds and caffeine. In embodiments, the anionicaromatic excipient compounds are metabolized in the body to yieldbiologically compatible byproducts.

c. Excipient Compound Category 3: Functionalized Amino Acids

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with one or more functionalized amino acids, where asingle functionalized amino acid or an oligopeptide comprising one ormore functionalized amino acids may be used as the excipient compound.In embodiments, the functionalized amino acid compounds comprisemolecules (“amino acid precursors”) that can be hydrolyzed ormetabolized to yield amino acids. In embodiments, the functionalizedamino acids can contain an aromatic functional group such as phenyl,benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromaticgroup, or a fused aromatic group. In embodiments, the functionalizedamino acid compounds can contain esterified amino acids, such as methyl,ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl,hydroxypropyl, PEG, and PPG esters. In embodiments, the functionalizedamino acid compounds are selected from the group consisting of arginineethyl ester, arginine methyl ester, arginine hydroxyethyl ester, andarginine hydroxypropyl ester. In embodiments, the functionalized aminoacid compound is a charged ionic compound in aqueous solution at neutralpH. For example, a single amino acid can be derivatized by forming anester, like an acetate or a benzoate, and the hydrolysis products wouldbe acetic acid or benzoic acid, both natural materials, plus the aminoacid. In embodiments, the functionalized amino acid excipient compoundsare metabolized in the body to yield biologically compatible byproducts.

d. Excipient Compound Category 4: Oligopeptides

High concentration solutions of therapeutic or non-therapeutic proteinscan be formulated with oligopeptides as excipient compounds. Inembodiments, the oligopeptide is designed such that the structure has acharged section and a bulky section. In embodiments, the oligopeptidesconsist of between 2 and 10 peptide subunits. The oligopeptide can bebi-functional, for example a cationic amino acid coupled to a non-polarone, or an anionic one coupled to a non-polar one. In embodiments, theoligopeptides consist of between 2 and 5 peptide subunits. Inembodiments, the oligopeptides are homopeptides such as polyglutamicacid, polyaspartic acid, poly-lysine, poly-arginine, and poly-histidine.In embodiments, the oligopeptides have a net cationic charge. In otherembodiments, the oligopeptides are heteropeptides, such as Trp2Lys3. Inembodiments, the oligopeptide can have an alternating structure such asan ABA repeating pattern. In embodiments, the oligopeptide can containboth anionic and cationic amino acids, for example, Arg-Glu. Withoutbeing bound by theory, the oligopeptides comprise structures that canassociate with proteins in such a way that it reduces the intermolecularinteractions that lead to high viscosity solutions; for example, theoligopeptide-protein association can be a charge-charge interaction,leaving a somewhat non-polar amino acid to disrupt hydrogen bonding ofthe hydration layer around the protein, thus lowering viscosity. In someembodiments, the oligopeptide excipient is present in the composition ina concentration of about 50 mg/ml or less.

e. Excipient Compound Category 5: Short-Chain Organic Acids

As used herein, the term “short-chain organic acids” refers to C2-C6organic acid compounds and the salts, esters, or lactones thereof. Thiscategory includes saturated and unsaturated carboxylic acids, hydroxyfunctionalized carboxylic acids, and linear, branched, or cycliccarboxylic acids. In embodiments, the acid group in the short-chainorganic acid is a carboxylic acid, sulfonic acid, phosphonic acid, or asalt thereof.

In addition to the four excipient categories above, high concentrationsolutions of therapeutic or non-therapeutic proteins can be formulatedwith short-chain organic acids, for example, the acid or salt forms ofsorbic acid, valeric acid, propionic acid, caproic acid, and ascorbicacid as excipient compounds. Examples of excipient compounds in thiscategory include potassium sorbate, taurine, calcium propionate,magnesium propionate, and sodium ascorbate.

f. Excipient Compound Category 6: Low Molecular Weight AliphaticPolyacids

High concentration solutions of therapeutic or non-therapeutic PEGylatedproteins can be formulated with certain excipient compounds that enablelower solution viscosity, where such excipient compounds are lowmolecular weight aliphatic polyacids. As used herein, the term “lowmolecular weight aliphatic polyacids” refers to organic aliphaticpolyacids having a molecular weight <about 1500, and having at least twoacidic groups, where an acidic group is understood to be aproton-donating moiety. Non-limiting examples of acidic groups includecarboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate, andnitrite groups. Acidic groups on the low molecular weight aliphaticpolyacid can be in the anionic salt form such as carboxylate,phosphonate, phosphate, sulfonate, sulfate, nitrate, and nitrite; theircounterions can be sodium, potassium, lithium, and ammonium. Specificexamples of low molecular weight aliphatic polyacids useful forinteracting with PEGylated proteins as described herein include maleicacid, tartaric acid, glutaric acid, malonic acid, citric acid,ethylenediaminetetraacetic acid (EDTA), aspartic acid, glutamic acid,alendronic acid, etidronic acid and salts thereof. Further examples oflow molecular weight aliphatic polyacids in their anionic salt forminclude phosphate (PO₄ ³⁻), hydrogen phosphate (HPO₄ ³⁻), dihydrogenphosphate (H₂PO₄ ⁻), sulfate (SO₄ ²⁻), bisulfate (HSO₄ ⁻), pyrophosphate(P₂O₇ ⁴⁻), carbonate (CO₃ ²⁻), and bicarbonate (HCO₃ ⁻). The counterionfor the anionic salts can be Na, Li, K, or ammonium ion. Theseexcipients can also be used in combination with excipients. As usedherein, the low molecular weight aliphatic polyacid can also be an alphahydroxy acid, where there is a hydroxyl group adjacent to a first acidicgroup, for example glycolic acid, lactic acid, and gluconic acid andsalts thereof. In embodiments, the low molecular weight aliphaticpolyacid is an oligomeric form that bears more than two acidic groups,for example polyacrylic acid, polyphosphates, polypeptides and saltsthereof. In some embodiments, the low molecular weight aliphaticpolyacid excipient is present in the composition in a concentration ofabout 50 mg/ml or less.

g. Excipient Compound Category 7: Diones and Sulfones

An effective viscosity-reducing excipient can be a molecule containing asulfone, sulfonamide, or dione functional group that is soluble in purewater to at least 1 g/L at 298K and having a net neutral charge at pH 7.Preferably, the molecule has a molecular weight of less than 1000 g/moland more preferably less than 500 g/mol. The diones and sulfoneseffective in reducing viscosity have multiple double bonds, are watersoluble, have no net charge at pH 7, and are not strong hydrogen bondingdonors. Not to be bound by theory, the double bond character can allowfor weak pi-stacking interactions with protein. In embodiments, at highprotein concentrations and in proteins that only develop high viscosityat high concentration, charged excipients are not effective becauseelectrostatic interaction is a longer-range interaction. Solvatedprotein surfaces are predominantly hydrophilic, making them watersoluble. The hydrophobic regions of proteins are generally shieldedwithin the 3-dimensional structure, but the structure is constantlyevolving, unfolding, and re-folding (sometimes called “breathing”) andthe hydrophobic regions of adjacent proteins can come into contact witheach other, leading to aggregation by hydrophobic interactions. Thepi-stacking feature of dione and sulfone excipients can mask hydrophobicpatches that may be exposed during such “breathing.” Another otherimportant role of the excipient can be to disrupt hydrophobicinteractions and hydrogen bonding between proteins in close proximity,which will effectively reduce solution viscosity. Dione and sulfonecompounds that fit this description include dimethylsulfone, ethylmethyl sulfone, ethyl methyl sulfonyl acetate, ethyl isopropyl sulfone,bis(methylsulfonyl)methane, methane sulfonamide, methionine sulfone,1,2-cyclopentanedione, 1,3-cyclopentanedione, 1,4-cyclopentanedione, andbutane-2,3-dione.

6. Protein/Excipient Solutions: Properties and Processes

In certain embodiments, solutions of therapeutic or non-therapeuticproteins formulated with above-identified excipient compounds orcombinations thereof (hereinafter, “excipient additives”), such ashindered amines, anionic aromatics, functionalized amino acids,oligopeptides, or short-chain organic acids, low molecular weightaliphatic polyacids, and diones and sulfones result in improvedprotein-protein interaction characteristics as measured by the proteindiffusion interaction parameter, kD, or the second virial coefficient,B22. As used herein, an “improvement” in one or more protein-proteininteraction parameters achieved by test formulations using theabove-identified excipient compounds or combinations thereof can referto a decrease in attractive protein-protein interactions when a testformulation is compared under comparable conditions with a comparableformulation that does not contain the excipient compounds or excipientadditives. Such improvements can be identified by measuring certainparameters that apply to the overall process or an aspect thereof, wherea parameter is any metric pertaining to the process where an alterationcan be can be quantified and compared to a previous state or to acontrol. A parameter can pertain to the process itself, such as itsefficiency, cost, yield, or rate.

A parameter can also be a proxy parameter that pertains to a feature oran aspect of the larger process. As an example, parameters such as thekD or B22 parameters can be termed proxy parameters. Measurements of kDand B22 can be made using standard techniques in the industry, and canbe an indicator of process-related parameters such as improved solutionproperties or stability of the protein in solution. Not to be bound bytheory, it is understood that a highly negative kD value can indicatethat the protein has strong attractive interactions, and this can leadto aggregation, instability, and rheology problems. When formulated inthe presence of certain of the above-identified excipient compounds orcombinations thereof, the same protein can have an improved proxyparameter of a less negative kD value, or a kD value near or above zero,with this improved proxy parameter being associated with an improvementin a process-related parameter.

In embodiments, certain of the above-described excipient compounds orcombinations thereof, such as hindered amines, anionic aromatics,functionalized amino acids, oligopeptides, short-chain organic acids,low molecular weight aliphatic polyacids, and/or diones and sulfones areused to improve a protein-related process, such as the manufacture,processing, sterile filling, purification, and analysis ofprotein-containing solutions, using processing methods such asfiltration, syringing, transferring, pumping, mixing, heating or coolingby heat transfer, gas transfer, centrifugation, chromatography, membraneseparation, centrifugal concentration, tangential flow filtration,radial flow filtration, axial flow filtration, lyophilization, and gelelectrophoresis. In these and related protein-related processes, theprotein of interest is dissolved in a solution that conveys it throughthe processing apparatus. Such solutions, referred to herein as “carriersolutions,” can include cell culture media (containing, for example,secreted proteins of interest), lysate solutions following the lysis ofhost cells (where the protein of interest resides in the lysate),elution solutions (which contain the protein of interest followingchromatographic separations), electrophoresis solutions, transportsolutions for carrying the protein of interest through conduits in aprocessing apparatus, and the like. A carrier solution containing theprotein of interest may also be termed a protein-containing solution ora protein solution. As described in more detail below, one or moreviscosity-reducing excipients can be added to the protein-containingsolution to improve various aspects of processing. As used herein, theterms “improve,” “improvements,” and the like refer to an advantageouschange in a parameter of interest in a carrier solution when thatparameter is compared to the same parameter as measured in a controlsolution. As used herein, a “control solution” means a solution thatlacks the viscosity-reducing excipient but otherwise substantiallysimilar to the carrier solution. As used herein, a “control process,”for example a control filtration process, a control chromatographicprocess, and the like, is a protein-related process that issubstantially similar to the protein-related process of interest and isperformed with a control solution instead of a carrier solution.

For example, in processes where a protein-containing solution is pumpedthrough conduits (e.g., flow chambers, piping or tubing), adding aviscosity-lowering excipient to the protein solution, as describedabove, before or during the pumping process can substantially reduce theforce and the power required to pump the solution. It is understood thatfluids generally exhibit a resistance to flow, i.e., a viscosity, andthat a force must be applied to the fluid to overcome this viscosity inorder to induce and propagate flow. The power, P, required for pumpingscales with the head, H, and capacity, Q, as shown in the followingequation:

P˜HQ  (Eq. 1)

Viscous fluids tend to increase the power requirements for pumps, tolower pump efficiency, to decrease pump head and capacity, and toincrease frictional resistance in piping. Adding the viscosity-loweringexcipients described above to a protein solution prior to or duringpumping can substantially lower processing costs by decreasing eitherthe head (H, Eq. 1) or the capacity (Q, Eq. 1) or both. The benefits ofreduced viscosity can be manifested, for example, by improvedthroughput, increased yield, or decreased processing time. Moreover,frictional losses from the transmission of a fluid through a conduit canaccount for a significant fraction of the costs associated withconveying such fluids. Adding a viscosity-lowering excipient asdescribed above to a protein solution prior to or during pumping cansubstantially lower processing costs by decreasing the frictionaccompanying the pumping process. Measurement of processing costsrepresents a processing parameter that can be improved by using aviscosity-reducing excipient.

These processes and processing methods for protein solutions can haveimproved efficiency due to the lower viscosity, improved solubility, orimproved stability of the proteins in the solution during manufacture,processing, purification, and analysis steps. Measurement of processingefficiency or measurement of proxy parameters such as viscosity,solubility or stability of the proteins in solution represent processingparameters that can be improved by using a viscosity-reducing excipient.Several different factors are understood to adversely affect proteinviscosity, solubility, and stability during processing. For example,protein-containing solutions are subject to a variety of physicalstressors during manufacturing and purification, including significantshear stresses induced by manipulating protein solutions through typicalprocessing operations, including, but not limited to, pumping, mixing,centrifugation, and filtration. In addition, during these processingsteps, air bubbles can become entrained within the fluid to whichproteins can adsorb. Such interfacial tension forces, coupled withtypical shear stresses encountered during processing, can cause adsorbedprotein molecules to unfold and aggregate. Additionally, significantprotein unfolding can occur during pump cavitation events and duringexposure to solid surfaces during manufacturing, such as ultrafiltrationand diafiltration membranes. Such events can impair protein folding andproduct quality.

For Newtonian fluids, the stress, τ, imposed by a given process scaleswith the shear rate, {dot over (γ)}, and viscosity of the fluid, η, asshown in the following equation:

τ={dot over (γ)}η  (Eq. 2)

By formulating a protein solution with one or more of theabove-described excipient compounds or combinations thereof, solutionviscosity is decreased, thus decreasing the shear stress encountered bythe protein solution. The decreased shear stress can improve thestability of the formulation being processed, as manifested, forexample, by a better or more desirable measurement of a processingparameter. Such improved processing parameters can include metrics suchas reduced levels of protein aggregates, particles, or subvisibleparticles (manifested macroscopically as turbidity), reduced productlosses, or improved overall yield. As another example of an improvedprocessing parameter, reducing viscosity of a protein-containingsolution can decrease the processing time for the solution. Theprocessing time for a given unit operation generally scales inverselywith the shear rate. Therefore, for a given characteristic stress, adecrease in protein solution viscosity by the addition of theabove-described excipient compounds or combinations thereof isassociated with an increase in shear rate ({dot over (γ)}, see Eq. 2),and therefore a decrease in the processing time.

During processing, it is understood that a protein in a solution may bea desired protein active ingredient, for example a therapeutic ornon-therapeutic protein. Facilitating the processing of such a proteinactive ingredient using the excipients described herein can increase theyield or the rate of production of the protein active ingredient, orimprove the efficiency of the particular process, or decrease the energyuse, or the like, any of which outcomes represent processing parametersthat have been improved by the use of the viscosity-reducing excipient.It is also understood that protein contaminants can be formed duringcertain processing technologies, for example during the fermentation andpurification steps of bioprocessing. Removing the contaminants morequickly, more thoroughly, or more efficiently can also improve theprocessing of the desired protein, i.e., the protein active ingredient;these outcomes represent processing parameters that have been improvedby the use of the viscosity-reducing excipient compound or additive. Asdescribed herein, certain excipients as described herein, by loweringsolution viscosity, improving protein stability, and/or increasingprotein solubility, can improve the transport of desired protein activeingredients, and can improve the removal of undesirable proteincontaminants; both effects, which represent processing parameters thathave been improved by the use of the viscosity-reducing excipient oradditive, show that these excipients or additives improve the overallprocess of protein manufacture.

Specific platform unit operation for therapeutic protein production andpurification offer further examples of the advantageous uses ofviscosity-reducing excipients as disclosed herein, and further examplesof these excipients' or additives' improving processing parameters. Forexample, introducing one or more of the viscosity-reducing excipientsdescribed above into these production and purification processes, asdescribed below, can provide substantial improvements in moleculestability and recovery, and a decrease in operation costs.

It is understood in the art that the widely-practiced technology forproducing and purifying therapeutic proteins like monoclonal antibodiesgenerally consists of a fermentation process followed by a series ofsteps for purification processing. Fermentation, or upstream processing(USP), comprises those steps by which therapeutic proteins are grown inbioreactors, typically using bacterial or mammalian cell lines. USP may,in embodiments, include steps such as those shown in FIG. 1 .Purification, or downstream processing (DSP) may, in embodiments,include steps such as those shown in FIG. 2 .

As shown in FIG. 1 , USP may commence with the step 102 of thawing ofvials from a master cell bank (MCB). The MCB can be expanded as shown instep 104, to form a working cell bank (not shown) and/or to produce theworking stock for further production. Cell culture takes place in aseries of seed and production bioreactors, as shown in steps 108 and110, to yield those bioreactor products 112 from which the desiredtherapeutic protein can be harvested, as shown in step 114. Followingharvest 114, the products can be submitted to further purification(i.e., DSP, as described below in more detail and as depicted in FIG. 2), or these products may be stored in bulk, typically by freezing andstoring at a temperature of approximately −80° C.

In embodiments, protein production by cell culture techniques can beimproved by the use of the above-identified excipients, as manifested byimprovements in process-related parameters. In embodiments, the desiredexcipient can be added during USP in an amount effective to reduce theviscosity of the cell culture medium by at least 20%. In otherembodiments, the desired excipient can be added during USP in an amounteffective to reduce the viscosity of the cell culture medium by at least30%. In embodiments, the desired excipient can be added to the cellculture medium in an amount of about 1 mM to about 400 mM. Inembodiments, the desired excipient can be added to the cell culturemedium in an amount of about 20 mM to about 200 mM. In embodiments, thedesired excipient can be added to the cell culture medium in an amountof about 25 mM to about 100 mM. The desired excipient or combination ofexcipients can be added directly to the cell culture medium, or it canbe added as a component of a more complex supplemental medium, forexample a nutrient-containing solution or “feed solution” that isformulated separately and added to the cell culture medium. Inembodiments, a second viscosity-reducing compound can be added to thecarrier solution, either directly or via a supplemental medium, whereinthe second viscosity-reducing compound adds an additional improvement toa particular parameter of interest.

As described below, there are many process-related parameters during USPthat can be improved by use of one or more viscosity-reducingexcipients. For example, in embodiments, use of a viscosity-reducingexcipient can improve parameters such as the rate and/or degree of cellgrowth during steps such as inoculum expansion 104, and cell culture 108and 110, and/or can improve proxy parameters that are correlated withthe improvement in various process parameters. For example, adding theabove-identified excipients to the USP process at a step such as theproduction bioreactor step 110, can decrease the viscosity of the cellculture medium, which can subsequently improve heat transfer efficiencyand gas transfer efficiency. Because the cell culture process requiresoxygen infusion to the cells to enable protein expression, and thediffusion of oxygen into the cells can therefore be a rate-limitingstep, improving the rate of oxygen uptake by improving gas transferefficiency through decreasing solution viscosity can improve the rate oramount of protein expression and/or its efficiency. In this context,parameters such as the rate of oxygen uptake and the rate of gastransfer efficiency can be deemed proxy parameters, whose improvement iscorrelated with an improvement in the process parameter of improvedprotein expression or improved processing efficiency. As anotherexample, the availability of viscosity-reducing excipients can improveprocessing, for example, during the inoculum expansion step 104 andduring the cell culture steps 108 and 110, by improving a proxyparameter such as the solubility of protein growth factors that arerequired for protein expression; with improved growth factor solubility,these substances can become more available to the cells, therebyfacilitating cell growth.

In embodiments, process parameters such as the amount of proteinrecovery or the rate of protein recovery during USP can be improved byreducing viscosity during USP by several mechanisms. For example, theharvest of therapeutic protein at the end of the lysis step duringharvest 114 from the completed cell culture can be more efficient or canbe otherwise improved with the use of the above-identified excipients.Not to be bound by theory, by reducing viscosity of the expressedprotein, these viscosity-reducing excipients can increase the efficiencyof diffusion of therapeutic protein away from other lysate components.In addition, the separation of membranes and other cell debris from theprotein-containing supernate can be accomplished with a fasterseparation rate or a higher degree of supernate purity, with the use ofthe viscosity-reducing excipients, thereby improving the processparameter of USP efficiency. Furthermore, the protein separation stepsthat use centrifugation or filtration steps can be accomplished fasterwith the use of the viscosity-reducing excipients, since the excipientsreduce the viscosity of the medium.

In embodiments, as an additional benefit, use of the above-describedviscosity-reducing excipients in cell culture can increase a processparameter such as protein yield during USP because protein misfoldingand aggregation are reduced. It is understood that, as the cell cultureis optimized to produce a maximum yield of recombinant protein, theresulting protein is expressed in a highly concentrated manner, whichcan result in misfolding; adding a viscosity-reducing excipient canreduce the attractive protein-protein interactions that lead tomisfolding and aggregation, thereby increasing the amount of intactrecombinant protein that is available for harvest 114.

Downstream processing (DSP), depicted in FIG. 2 in an illustrativeembodiment, involves a sequence of steps that results in the recoveryand purification of therapeutic proteins, for example monoclonalantibodies, biopharmaceuticals, vaccines, and other biologics. At theend of USP, the therapeutic protein of interest can be dissolved in thecell culture medium, having been secreted from the host cells. Thetherapeutic protein can also be dissolved in a fluid medium followingthe lysis of the host cells at the end of the USP sequences. DSP isundertaken to retrieve the protein of interest from the solution inwhich it is dissolved (e.g., the culture medium or host cell lysatemedium), and to purify it. During DSP, (i) various contaminants (such asinsoluble cell debris and particulates) are removed from the media, (ii)the protein product is isolated through techniques such as extraction,precipitation, adsorption or ultrafiltration, (iii) the protein productis purified through techniques such as affinity chromatography,precipitation, or crystallization, and (iv) the product is furtherpolished, and viruses are removed.

As shown in FIG. 2 , a feedstock from cell culture harvest 200 (also asdescribed in FIG. 1 ) is initially subjected to affinity chromatography204, typically involving Protein-A chromatography or other analogouschromatographic steps. The virus inactivation step 208 typically entailssubjecting the feedstock to a low pH hold. One or more polishingchromatography steps 210 and 212 are performed to remove impurities,such as host cell proteins (HCP), DNA, charged variants, and aggregates.Cation exchange (CEX) chromatography is commonly used as an initialpolishing chromatography step 210, but it may be accompanied by a secondchromatography step 212 that either precedes or follows it. The secondchromatography step 212 further removes host-cell-related impurities(e.g., HCP or DNA), or product related impurities such as aggregates.Anion exchange (AEX) chromatography and hydrophobic interactionchromatography (HIC) can be employed as second chromatography steps 212.Virus filtration 214 is performed to effect virus removal. Finalpurification steps 218 can include ultrafiltration and diafiltration,and preparation for formulation.

As generally described above, purification processes or DSP followingthe fermentation process can include (1) cell culture harvest, (2)chromatography (e.g., Protein-A chromatography and chromatographicpolishing steps, including ion exchange and hydrophobic interactionchromatography), (3) viral inactivation, and (4) filtration (e.g., viralfiltration, sterile filtration, dialysis, and ultrafiltration anddiafiltration steps to concentrate the protein and exchange the proteininto the formulation buffer). Examples are provided below to illustratethe advantages from using a viscosity-reducing excipient as describedherein to improve process parameters associated with these purificationprocesses. It is understood that the viscosity-reducing excipient orcombinations thereof can be introduced at any phase of DSP by adding itto a carrier solution or in any other way engineering the contact of theprotein of interest with the excipient, whether in soluble or stabilizedform. In embodiments, a second viscosity-reducing compound can be addedto the carrier solution during DSP, wherein the secondviscosity-reducing compound adds an additional improvement to aparticular parameter of interest.

(1) Cell culture harvest: Cell culture harvest generally involvescentrifugation and depth filtration operations in which cellular debrisis physically removed from protein-containing solutions. Thecentrifugation step can provide a more complete separation of solubleprotein from cell debris with the benefit of a viscosity-reducingexcipient. Whether done by batch or continuous processing, thecentrifuge separation requires the dense phase to consolidate as much aspossible to maximize recovery of the target protein. In embodiments,addition of the above-identified excipients or combinations thereof canincrease the process parameter of protein yield, for example, byincreasing the yield of protein-containing centrate that flows away fromthe dense phase of the centrifuge separation process. The depthfiltration step is a viscosity-limited step, and thus can be made moreefficient by using an excipient that reduces solution viscosity. Theseprocesses can also introduce air bubbles into the protein solution,which can couple with shear-induced stresses to destabilize thetherapeutic protein molecules being purified. Adding aviscosity-reducing excipient to the protein-containing solution, beforeand/or during cell culture harvest, as described above, can protect theprotein from these stresses, thereby improving the process parameter ofquantified product recovery.

(2) Chromatography: After cell culture harvest by centrifugation orfiltration, chromatography is typically used to separate the therapeuticprotein from the fermentation broth. Protein A chromatography is usedwhen the therapeutic protein is an antibody: Protein A is selectivetowards IgG antibodies, which it will bind dynamically at a high flowrate and capacity. Cation exchange (CEX) chromatography can be used as acost-effective alternative to Protein A chromatography. If CEX is used,the pH of the feed must be adjusted and its conductivity decreased priorto loading onto the column to optimize the dynamic binding capacity.Mimetic resins can also be used as an alternative to Protein Achromatography. These resins provide ligands to bind immunoglobulins,for example Ig-binding proteins like protein G or protein L, syntheticligands, or protein A-like porous polymers.

Other chromatography processes can be employed during DSP. Ion exchangechromatography (IEC) can be used to remove impurities introduced duringprevious processes, for example, leached Protein A, endotoxins orviruses from the cell line, remaining host cell proteins or DNA, ormedia components. IEC, whether CEX or anion exchange chromatography, canbe applied directly after Protein A chromatography. Hydrophobicinteraction chromatography (HIC) can complement IEC, generally used as apolishing step to remove aggregates. In embodiments, the use of theabove-identified excipients can increase the solubility of, and decreasethe viscosity of host cell proteins during chromatography column loadingsteps. In embodiments, the use of the above-identified excipients canincrease the solubility of, and decrease the viscosity of thetherapeutic protein during chromatography column loading steps andelution steps.

Chromatographic processes during protein purification impose harshconditions on the protein formulation, such as (a) low pH conditionsduring elution from Protein-A chromatography columns, (b) elevated localprotein concentration (often on the order of 300-400 mg/mL) within thepore-space of chromatographic resin, (c) elevated salt concentrationsduring ion exchange chromatography, and (d) elevated concentrations ofsalting-out agents during elution from HIC columns. Adding aviscosity-reducing excipient to the protein-containing solution, beforeand/or during chromatography, as described above, can facilitate thetransit of the proteins through the chromatography column so that theyare less exposed to the potentially damaging conditions imposed bychromatographic processing steps. In addition, the elevated localprotein concentration within the column pore-space can result in ahighly viscous material within this space, which places significant backpressure on the column. To alleviate this back pressure, media withrelatively large pores are typically used. However, the resolving powerof large-pore media is lower than small-pore counterparts. Theincorporation of viscosity-modifying excipients as described above canenable the use of smaller pores in the chromatographic media. Inembodiments, the elution steps from Protein-A chromatography expose thetherapeutic protein to a low pH condition that can reduce solubility andincrease aggregation of the target protein; addition of the excipientscan increase the solubility of the target protein such that recoveryyield from the Protein-A chromatography step is improved. In otherembodiments, use of the excipient can enable elution of the targetprotein from Protein-A resin at a higher pH, and this can reducechemical stresses on the target protein, resulting in improving aprocess parameter of protein yield by reducing the amount of proteindegradation during processing.

(3) Viral inactivation: Viral inactivation processes typically involveholding the protein solution at a low pH, e.g., pH lower than 4, for anextended period of time. This environment, though, can destabilizetherapeutic proteins. Formulating the protein in the presence of aviscosity-reducing excipient, for example, by adding aviscosity-reducing excipient before and/or during a viral inactivationprocess, can improve process parameters such as the stability orsolubility of the protein, or its net yield.

(4) Filtration: Filtration processes include viral filtration processes(nanofiltration) to remove virus particles, andultrafiltration/diafiltration processes to concentrate protein solutionsand to exchange buffer systems.

(a) Viral filtration purifies the protein solution by removing virusparticles, which can be on the order of twice the size of a recombinanthuman monoclonal antibodies. Thus, the filtration membrane for viralfiltration can require nano-sized pores. As a result of the small poresize through which the proteins must pass, this filtration step canintroduce stress to the protein, and is accompanied by significantlevels of membrane fouling from protein aggregate particles. Theaddition of a viscosity-reducing excipient, for example, before and/orduring filtration, as described above, can reduce a measurable parametersuch as back pressure in the filtration system by increasing collectivediffusivity, and can decrease the tendency for membrane fouling bymitigating the protein-protein interactions that give rise to it. Theend result is improvement in those parameters indicting improvedperformance of the viral filtration unit during protein purification.

(b) Ultrafiltration and diafiltration (UF/DF) processes concentrateprotein solutions and exchange buffer systems by passing theprotein-containing solution through a filter membrane with acharacteristic molecular weight cutoff that is smaller than the proteinof interest. In this step, the protein solution faces high shearstresses within the filter units, elevated protein concentrations, andadsorption of the protein to the hydrophobic membranes typically usedduring UF/DF processes, all of which can increase protein aggregation.The addition of a viscosity-reducing excipient, for example, beforeand/or during a UF/DF process, as described above, can reduce backpressure in the filtration system by increasing collective diffusivity(measured, for example, by an increase in k_(D)). This not only reducesshear stress across the membrane, but also promotes back-diffusion awayfrom the filter membrane, thus lowering the effective proteinconcentration at the membrane interface and increasing the permeateflux. As a result, the use of viscosity-reducing excipients can improveparameters associated with higher throughput during these filtrationprocesses, with reduced product losses and increased net yield.Additionally, passing viscous fluids through ultra- and diafilters canproduce a large pressure drop across the filter device, making theseparation inefficient. Formulating the protein solution in the presenceof viscosity-reducing excipients as described above can substantiallyreduce the pressure drop across the filter device, thereby improving theprocess parameters of operation costs and processing time by decreasingthem both.

After the upstream protein processing or downstream purification havebeen completed with the added excipient, the excipient can remain as apart of the drug substance mixture or it can be separated from theprotein active ingredient. Typical small molecule separation methods canbe used to separate the excipient from the protein active ingredient,such as buffer exchange, ion exchange, ultrafiltration, and dialysis. Inaddition to the beneficial effects on the protein purification processesas outlined above, the use of the above-identified excipients canprotect and preserve equipment used in protein manufacture, processing,and purification. For example, equipment-related processes such as thecleanup, sterilization, and maintenance of protein processing equipmentcan be facilitated by the use of the above-identified excipients due todecreased fouling, decreased denaturing, lower viscosity, and improvedsolubility of the protein, and parameters associated with theimprovement of these processes are similarly improved.

While the use of an excipient compound to improve upstream and/ordownstream processing has been described extensively herein, it isunderstood that a combination of excipients can be added together inorder to achieve a desired effect, such as an improvement in a parameterof interest. The term “excipient additive” can refer to either a singleexcipient compound that leads to the desired effect or improvedparameter, or to a combination of excipient compounds where thecombination is responsible for the desired effect or the improvedparameter.

EXAMPLES

Materials:

-   -   Bovine gamma globulin (BGG), >99% purity, Catalog #G5009, Sigma        Aldrich    -   Histidine, Sigma Aldrich    -   Other materials described in the examples below were from Sigma        Aldrich unless otherwise specified.

Example 1: Preparation of Formulations Containing Excipient Compoundsand Test Protein

Formulations were prepared using an excipient compound and a testprotein, where the test protein was intended to simulate either atherapeutic protein that would be used in a therapeutic formulation, ora non-therapeutic protein that would be used in a non-therapeuticformulation. Such formulations were prepared in 50 mM histidinehydrochloride with different excipient compounds for viscositymeasurement in the following way. Histidine hydrochloride was firstprepared by dissolving 1.94 g histidine in distilled water and adjustingthe pH to about 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St.Louis, Mo.) and then diluting to a final volume of 250 mL with distilledwater in a volumetric flask. Excipient compounds were then dissolved in50 mM histidine HCl. Lists of excipients are provided below in Examples4, 5, 6, and 7. In some cases excipient compounds were adjusted to pH 6prior to dissolving in 50 mM histidine HCl. In this case the excipientcompounds were first dissolved in deionized water at about 5 wt % andthe pH was adjusted to about 6.0 with either hydrochloric acid or sodiumhydroxide. The prepared salt solution was then placed in a convectionlaboratory oven at about 65° C. to evaporate the water and isolate thesolid excipient. Once excipient solutions in 50 mM histidine HCl hadbeen prepared, the test protein bovine gamma globulin (BGG) wasdissolved at a ratio of about 0.336 g BGG per 1 mL excipient solution.This resulted in a final protein concentration of about 280 mg/mL.Solutions of BGG in 50 mM histidine HCl with excipient were formulatedin 20 mL vials and allowed to shake at 100 rpm on an orbital shakertable overnight. BGG solutions were then transferred to 2 mLmicrocentrifuge tubes and centrifuged for ten minutes at 2300 rpm in anIEC MicroMax microcentrifuge to remove entrained air prior to viscositymeasurement.

Example 2: Viscosity Measurement

Viscosity measurements of formulations prepared as described in Example1 were made with a DV-IIT LV cone and plate viscometer (BrookfieldEngineering, Middleboro, Mass.). The viscometer was equipped with aCP-40 cone and was operated at 3 rpm and 25° C. The formulation wasloaded into the viscometer at a volume of 0.5 mL and allowed to incubateat the given shear rate and temperature for 3 minutes, followed by ameasurement collection period of twenty seconds. This was then followedby 2 additional steps consisting of 1 minute of shear incubation andsubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample.

Example 3: Protein Concentration Measurement

The concentration of the protein in the experimental solutions wasdetermined by measuring the optical absorbance of the protein solutionat a wavelength of 280 nm in a UV/VIS Spectrometer (Perkin Elmer Lambda35). First the instrument was calibrated to zero absorbance with a 50 mMhistidine buffer at pH 6. Next the protein solutions were diluted by afactor of 300 with the same histidine buffer and the absorbance at 280nm recorded. The final concentration of the protein in the solution wascalculated by using the extinction coefficient value of 1.264mL/(mg×cm).

Example 4: Formulations with Hindered Amine Excipient Compounds

Formulations containing 280 mg/mL BGG were prepared as described inExample 1, with some samples containing added excipient compounds. Inthese tests, the hydrochloride salts of dimethylcyclohexylamine (DMCHA),dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA),triethanolamine (TEA), dimethylethanolamine (DMEA), and niacinamide weretested as examples of the hindered amine excipient compounds. Also, ahydroxybenzoic acid salt of DMCHA and a taurine-dicyandiamide adductwere tested as examples of the hindered amine excipient compounds. Theviscosity of each protein solution was measured as described in Example2, and the results are presented in Table 1 below, showing the benefitof the added excipient compounds in reducing viscosity.

TABLE 1 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 4.1 None 0 79  0% 4.2 DMCHA-HCl28 50 37% 4.3 DMCHA-HCl 41 43 46% 4.4 DMCHA-HCl 50 45 43% 4.5 DMCHA-HCl82 36 54% 4.6 DMCHA-HCl 123 35 56% 4.7 DMCHA-HCl 164 40 49% 4.8DMAPA-HCl 87 57 28% 4.9 DMAPA-HCl 40 54 32% 4.10 DCHMA-HCl 29 51 35%4.11 DCHMA-HCl 50 51 35% 4.14 TEA-HCl 97 51 35% 4.15 TEA-HCl 38 57 28%4.16 DMEA-HCl 51 51 35% 4.17 DMEA-HCl 98 47 41% 4.20 DMCHA- 67 46 42%hydroxybenzoate 4.21 DMCHA- 92 42 47% hydroxybenzoate 4.22 Product ofExample 8 26 58 27% 4.23 Product of Example 8 58 50 37% 4.24 Product ofExample 8 76 49 38% 4.25 Product of Example 8 103 46 42% 4.26 Product ofExample 8 129 47 41% 4.27 Product of Example 8 159 42 47% 4.28 Productof Example 8 163 42 47% 4.29 Niacinamide 48 39 51% 4.30 N-Methyl-2- 3045 43% pyrrolidone 4.31 N-Methyl-2- 52 52 34% pyrrolidone

Example 5: Formulations with Anionic Aromatic Excipient Compounds

Formulations of 280 mg/mL BGG were prepared as described in Example 1,with some samples containing added excipient compounds. The viscosity ofeach solution was measured as described in Example 2, and the resultsare presented in Table 2 below, showing the benefit of the addedexcipient compounds in reducing viscosity.

TABLE 2 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 5.1 None 0 79  0% 5.2 Sodiumaminobenzoate 43 48 39% 5.3 Sodium 26 50 37% hydroxybenzoate 5.4 Sodiumsulfanilate 44 49 38% 5.5 Sodium sulfanilate 96 42 47% 5.6 Sodium indoleacetate 52 58 27% 5.7 Sodium indole acetate 27 78  1% 5.8 Vanillic acid,sodium 25 56 29% salt 5.9 Vanillic acid, sodium 50 50 37% salt 5.10Sodium salicylate 25 57 28% 5.11 Sodium salicylate 50 52 34% 5.12Adenosine 26 47 41% monophosphate 5.13 Adenosine 50 66 16% monophosphate5.14 Sodium benzoate 31 61 23% 5.15 Sodium benzoate 56 62 22%

Example 6: Formulations with Oligopeptide Excipient Compounds

Oligopeptides (n=5) were synthesized by NeoBioLab Inc. (Woburn, Mass.)in >95% purity with the N terminus as a free amine and the C terminus asa free acid. Dipeptides (n=2) were synthesized by LifeTein LLC(Somerset, N.J.) in 95% purity. Formulations of 280 mg/mL BGG wereprepared as described in Example 1, with some samples containing thesynthetic oligopeptides as added excipient compounds. The viscosity ofeach solution was measured as described in Example 2, and the resultsare presented in Table 3 below, showing the benefit of the addedexcipient compounds in reducing viscosity.

TABLE 3 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 6.1 None 0 79  0% 6.2 ArgX5 10055 30% 6.3 ArgX5 50 54 32% 6.4 HisX5 100 62 22% 6.5 HisX5 50 51 35% 6.6HisX5 25 60 24% 6.7 Trp2Lys3 100 59 25% 6.8 Trp2Lys3 50 60 24% 6.9 AspX5100 102 −29%   6.10 AspX5 50 82  −4%   6.11 Dipeptide LE 50 72  9%(Leu-Glu) 6.12 Dipeptide YE 50 55 30% (Tyr-Glu) 6.13 Dipeptide RP 50 5135% (Arg-Pro) 6.14 Dipeptide RK 50 53 33% (Arg-Lys) 6.15 Dipeptide RH 5052 34% (Arg-His) 6.16 Dipeptide RR 50 57 28% (Arg-Arg) 6.17 Dipeptide RE50 50 37% (Arg-Glu) 6.18 Dipeptide LE 100 87 −10%   (Leu-Glu) 6.19Dipeptide YE 100 68 14% (Tyr-Glu) 6.20 Dipeptide RP 100 53 33% (Arg-Pro)6.21 Dipeptide RK 100 64 19% (Arg-Lys) 6.22 Dipeptide RH 100 72  9%(Arg-His) 6.23 Dipeptide RR 100 62 22% (Arg-Arg) 6.24 Dipeptide RE 10066 16% (Arg-Glu)

Example 8: Synthesis of Guanyl Taurine Excipient

Guanyl taurine was prepared following method described in U.S. Pat. No.2,230,965. Taurine (Sigma-Aldrich, St. Louis, Mo.) 3.53 parts were mixedwith 1.42 parts of dicyandiamide (Sigma-Aldrich, St. Louis, Mo.) andgrinded in a mortar and pestle until a homogeneous mixture was obtained.Next the mixture was placed in a flask and heated at 200° C. for 4hours. The product was used without further purification.

Example 9: Protein Formulations Containing Excipient Compounds

Formulations were prepared using an excipient compound and a testprotein, where the test protein was intended to simulate either atherapeutic protein that would be used in a therapeutic formulation, ora non-therapeutic protein that would be used in a non-therapeuticformulation. Such formulations were prepared in 50 mM aqueous histidinehydrochloride buffer solution with different excipient compounds forviscosity measurement in the following way. Histidine hydrochloridebuffer solution was first prepared by dissolving 1.94 g histidine indistilled water and adjusting the pH to about 6.0 with 1 M hydrochloricacid (Sigma-Aldrich, St. Louis, Mo.) and then diluting to a final volumeof 250 mL with distilled water in a volumetric flask. Excipientcompounds were then dissolved in the 50 mM histidine HCl buffersolution. A list of the excipient compounds is provided in Table 4. Insome cases, excipient compounds were dissolved in 50 mM histidine HClbuffer solution and the resulting solution pH was adjusted with smallamounts of sodium hydroxide or hydrochloric acid to achieve pH 6 priorto dissolution of the model protein. In some cases, excipient compoundswere adjusted to pH 6 prior to dissolving in 50 mM histidine HCl. Inthis case the excipient compounds were first dissolved in deionizedwater at about 5 wt % and the pH was adjusted to about 6.0 with eitherhydrochloric acid or sodium hydroxide. The prepared salt solution wasthen placed in a convection laboratory oven at about 65° C. to evaporatethe water and isolate the solid excipient. Once excipient solutions in50 mM histidine HCl had been prepared, the test protein, bovine gammaglobulin (BGG) was dissolved at a ratio to achieve a final proteinconcentration of about 280 mg/mL. Solutions of BGG in 50 mM histidineHCl with excipient were formulated in 20 mL vials and allowed to shakeat 100 rpm on an orbital shaker table overnight. BGG solutions were thentransferred to 2 mL microcentrifuge tubes and centrifuged for tenminutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25° C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation andsubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient. Thenormalized viscosity is the ratio of the viscosity of the model proteinsolution with excipient to the viscosity of the model protein solutionwith no excipient.

TABLE 4 Excipient Normalized Test Concentration Viscosity ViscosityNumber Excipient Added (mg/mL) (cP) Reduction 9.1 DMCHA-HCl 120 0.44 56%9.2 Niacinamide 50 0.51 49% 9.3 Isonicotinamide 50 0.48 52% 9.4 TyramineHCl 70 0.41 59% 9.5 Histamine HCl 50 0.41 59% 9.6 Imidazole HCl 100 0.4357% 9.7 2-methyl-2- 60 0.43 57% imidazoline HCl 9.8 1-butyl-3- 100 0.4852% methylimidazolium chloride 9.9 Procaine HCl 50 0.53 47% 9.103-aminopyridine 50 0.51 49% 9.11 2,4,6- 50 0.49 51% trimethylpyridine9.12 3-pyridine 50 0.53 47% methanol 9.13 Nicotinamide 20 0.56 44%adenine dinucleotide 9.15 Sodium 55 0.57 43% phenylpyruvate 9.162-Pyrrolidinone 60 0.68 32% 9.17 Morpholine HCl 50 0.60 40% 9.18Agmatine sulfate 55 0.77 23% 9.19 1-butyl-3- 60 0.66 34%methylimidazolium iodide 9.21 L-Anserine nitrate 50 0.79 21% 9.221-hexyl-3- 65 0.89 11% methylimidazolium chloride 9.23 N,N-diethyl 500.67 33% nicotinamide 9.24 Nicotinic acid, 100 0.54 46% sodium salt 9.25Biotin 20 0.69 31%

Example 10: Preparation of Formulations Containing ExcipientCombinations and Test Protein

Formulations were prepared using a primary excipient compound, asecondary excipient compound and a test protein, where the test proteinwas intended to simulate either a therapeutic protein that would be usedin a therapeutic formulation, or a non-therapeutic protein that would beused in a non-therapeutic formulation. The primary excipient compoundswere selected from compounds having both anionic and aromaticfunctionality, as listed below in Table 5. The secondary excipientcompounds were selected from compounds having either nonionic orcationic charge at pH 6 and either imidazoline or benzene rings, aslisted below in Table 5. Formulations of these excipients were preparedin 50 mM histidine hydrochloride buffer solution for viscositymeasurement in the following way. Histidine hydrochloride was firstprepared by dissolving 1.94 g histidine in distilled water and adjustingthe pH to about 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St.Louis, Mo.) and then diluting to a final volume of 250 mL with distilledwater in a volumetric flask. The individual primary or secondaryexcipient compounds were then dissolved in 50 mM histidine HCl.Combinations of primary and secondary excipients were dissolved in 50 mMhistidine HCl and the resulting solution pH adjusted with small amountsof sodium hydroxide or hydrochloric acid to achieve pH 6 prior todissolution of the model protein. Once excipient solutions had beenprepared as described above, the test protein bovine gamma globulin(BGG) was dissolved into each test solution at a ratio to achieve afinal protein concentration of about 280 mg/mL. Solutions of BGG in 50mM histidine HCl with excipient were formulated in 20 mL vials andallowed to shake at 100 rpm on an orbital shaker table overnight. BGGsolutions were then transferred to 2 mL microcentrifuge tubes andcentrifuged for ten minutes at 2300 rpm in an IEC MicroMaxmicrocentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25° C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation and asubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, andsummarized in Table 5 below. The normalized viscosity is the ratio ofthe viscosity of the model protein solution with excipient to theviscosity of the model protein solution with no excipient. The exampleshows that a combination of primary and secondary excipients can give abetter result than a single excipient.

TABLE 5 Primary Excipient Secondary Excipient Test ConcentrationConcentration Normalized Number Name (mg/mL) Name (mg/mL) Viscosity 10.1Salicylic Acid 30 None 0 0.79 10.2 Salicylic Acid 25 Imidazole 4 0.5910.3 4-hydroxybenzoic 30 None 0 0.61 acid 10.4 4-hydroxybenzoic 25Imidazole 5 0.57 acid 10.5 4-hydroxybenzene 31 None 0 0.59 sulfonic acid10.6 4-hydroxybenzene 26 Imidazole 5 0.70 sulfonic acid 10.74-hydroxybenzene 25 Caffeine 5 0.69 sulfonic acid 10.8 None 0 Caffeine10 0.73 10.9 None 0 Imidazole 5 0.75

Example 11: Preparation of Formulations Containing ExcipientCombinations and Test Protein

Formulations were prepared using a primary excipient compound, asecondary excipient compound and a test protein, where the test proteinwas intended to simulate a therapeutic protein that would be used in atherapeutic formulation, or a non-therapeutic protein that would be usedin a non-therapeutic formulation. The primary excipient compounds wereselected from compounds having both anionic and aromatic functionality,as listed below in Table 6. The secondary excipient compounds wereselected from compounds having either nonionic or cationic charge at pH6 and either imidazoline or benzene rings, as listed below in Table 6.Formulations of these excipients were prepared in distilled water forviscosity measurement in the following way. Combinations of primary andsecondary excipients were dissolved in distilled water and the resultingsolution pH adjusted with small amounts of sodium hydroxide orhydrochloric acid to achieve pH 6 prior to dissolution of the modelprotein. Once excipient solutions in distilled water had been prepared,the test protein bovine gamma globulin (BGG) was dissolved at a ratio toachieve a final protein concentration of about 280 mg/mL. Solutions ofBGG in distilled water with excipient were formulated in 20 mL vials andallowed to shake at 100 rpm on an orbital shaker table overnight. BGGsolutions were then transferred to 2 mL microcentrifuge tubes andcentrifuged for ten minutes at 2300 rpm in an IEC MicroMaxmicrocentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25° C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation and asubsequent twenty-second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, andsummarized in Table 6 below. The normalized viscosity is the ratio ofthe viscosity of the model protein solution with excipient to theviscosity of the model protein solution with no excipient. The exampleshows that a combination of primary and secondary excipients can give abetter result than a single excipient.

TABLE 6 Primary Excipient Secondary Excipient Test ConcentrationConcentration Normalized Number Name (mg/mL) Name (mg/mL) Viscosity 11.1Salicylic Acid 20 None 0 0.96 11.2 Salicylic Acid 20 Caffeine 5 0.7111.3 Salicylic Acid 20 Niacinamide 5 0.76 11.4 Salicylic Acid 20Imidazole 5 0.73

Example 12: Preparation of Formulations Containing Excipient Compoundsand PEG

Materials: All materials were purchased from Sigma-Aldrich, St. Louis,Mo. Formulations were prepared using an excipient compound and PEG,where the PEG was intended to simulate a therapeutic PEGylated proteinthat would be used in a therapeutic formulation. Such formulations wereprepared by mixing equal volumes of a solution of PEG with a solution ofthe excipient. Both solutions were prepared in a Tris buffer consistingof 10 mM Tris, 135 mM NaCl, 1 mM trans-cinnamic acid at pH of 7.3.

The PEG solution was prepared by mixing 3 g of poly(ethylene oxide)average Mw ˜1,000,000 (Aldrich Catalog #372781) with 97 g of the Trisbuffer solution. The mixture was stirred overnight for completedissolution.

An example of the excipient solution preparation is as follows: Anapproximately 80 mg/mL solution of citric acid in the Tris buffer wasprepared by dissolving 0.4 g of citric acid (Aldrich cat. #251275) in 5mL of the Tris buffer solution and adjusted the pH to 7.3 with minimumamount of 10 M NaOH solution.

The PEG excipient solution was prepared by mixing 0.5 mL of the PEGsolution with 0.5 mL of the excipient solution and mixed by using avortex for a few seconds. A control sample was prepared by mixing 0.5 mLof the PEG solution with 0.5 mL of the Tris buffer solution.

Example 13: Viscosity Measurements of Formulations Containing ExcipientCompounds and PEG

Viscosity measurements of the formulations prepared were made with aDV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro,Mass.). The viscometer was equipped with a CP-40 cone and was operatedat 3 rpm and 25° C. The formulation was loaded into the viscometer at avolume of 0.5 mL and allowed to incubate at the given shear rate andtemperature for 3 minutes, followed by a measurement collection periodof twenty seconds. This was then followed by 2 additional stepsconsisting of 1 minute of shear incubation and subsequent twenty secondmeasurement collection period. The three data points collected were thenaveraged and recorded as the viscosity for the sample.

The results presented in Table 7 show the effect of the added excipientcompounds in reducing viscosity.

TABLE 7 Excipient Test Concentration Viscosity Viscosity NumberExcipient (mg/mL) (cP) Reduction 13.1 None 0 104.8  0% 13.2 Citric acidNa salt 40 56.8 44% 13.3 Citric acid Na salt 20 73.3 28% 13.4 glycerolphosphate 40 71.7 30% 13.5 glycerol phosphate 20 83.9 18% 13.6 Ethylenediamine 40 84.7 17% 13.7 Ethylene diamine 20 83.9 15% 13.8 EDTA/K salt40 67.1 36% 13.9 EDTA/K salt 20 76.9 27% 13.10 EDTA/Na salt 40 68.1 35%13.11 EDTA/Na salt 20 77.4 26% 13.12 D-Gluconic acid/K salt 40 80.32 23%13.13 D-Gluconic acid/K salt 20 88.4 16% 13.14 D-Gluconic acid/Na salt40 81.24 23% 13.15 D-Gluconic acid/Na salt 20 86.6 17% 13.16 lacticacid/K salt 40 80.42 23% 13.17 lactic acid/K salt 20 85.1 19% 13.18lactic acid/Na salt 40 86.55 17% 13.19 lactic acid/Na salt 20 87.2 17%13.20 etidronic acid/K salt 24 71.91 31% 13.21 etidronic acid/K salt 1280.5 23% 13.22 etidronic acid/Na salt 24 71.6 32% 13.23 etidronicacid/Na salt 12 79.4 24%

Example 14: Preparation of PEGylated BSA with 1 PEG Chain Per BSAMolecule

To a beaker was added 200 mL of a phosphate buffered saline (AldrichCat. #P4417) and 4 g of BSA (Aldrich Cat. #A7906) and mixed with amagnetic bar. Next 400 mg of methoxy polyethylene glycol maleimide,MW=5,000, (Aldrich Cat. #63187) was added. The reaction mixture wasallowed to react overnight at room temperature. The following day, 20drops of HCl 0.1 M were added to stop the reaction. The reaction productwas characterized by SDS-Page and SEC which clearly showed the PEGylatedBSA. The reaction mixture was placed in an Amicon centrifuge tube with amolecular weight cutoff (MWCO) of 30,000 and concentrated to a fewmilliliters. Next the sample was diluted 20 times with a histidinebuffer, 50 mM at a pH of approximately 6, followed by concentratinguntil a high viscosity fluid was obtained. The final concentration ofthe protein solution was obtained by measuring the absorbance at 280 nmand using a coefficient of extinction for the BSA of 0.6678. The resultsindicated that the final concentration of BSA in the solution was 342mg/mL.

Example 15: Preparation of PEGylated BSA with Multiple PEG Chains PerBSA Molecule

A 5 mg/mL solution of BSA (Aldrich A7906) in phosphate buffer, 25 mM atpH of 7.2, was prepared by mixing 0.5 g of the BSA with 100 mL of thebuffer. Next 1 g of a methoxy PEG propionaldehyde Mw=20,000 (JenKemTechnology, Plano, Tex. 75024) was added followed by 0.12 g of sodiumcyanoborohydride (Aldrich 156159). The reaction was allowed to proceedovernight at room temperature. The following day the reaction mixturewas diluted 13 times with a Tris buffer (10 mM Tris, 135 mM NaCl atpH=7.3) and concentrated using Amicon centrifuge tubes MWCO of 30,000until a concentration of approximately 150 mg/mL was reached.

Example 16: Preparation of PEGylated Lysozyme with Multiple PEG ChainsPer Lysozyme Molecule

A 5 mg/mL solution of lysozyme (Aldrich L6876) in phosphate buffer, 25mM at pH of 7.2, was prepared by mixing 0.5 g of the lysozyme with 100mL of the buffer. Next 1 g of a methoxy PEG propionaldehyde Mw=5,000(JenKem Technology, Plano, Tex. 75024) was added followed by 0.12 g ofsodium cyanoborohydride (Aldrich 156159). The reaction was allowed toproceed overnight at room temperature. The following day the reactionmixture was diluted 49 times with the phosphate buffer, 25 mM at pH of7.2, and concentrated using Amicon centrifuge tubes MWCO of 30,000. Thefinal concentration of the protein solution was obtained by measuringthe absorbance at 280 nm and using a coefficient of extinction for thelysozyme of 2.63. The final concentration of lysozyme in the solutionwas 140 mg/mL.

Example 17: Effect of Excipients on Viscosity of PEGylated BSA with 1PEG Chain Per BSA Molecule

Formulations of PEGylated BSA (from Example 14 above) with excipientswere prepared by adding 6 or 12 milligrams of the excipient salt to 0.3mL of the PEGylated BSA solution. The solution was mixed by gentlyshaking and the viscosity was measured by a RheoSense microVisc equippedwith an A10 channel (100-micron depth) at a shear rate of 500 sec-1. Theviscometer measurements were completed at ambient temperature.

The results presented in Table 8 shows the effect of the added excipientcompounds in reducing viscosity.

TABLE 8 Excipient Test Concentration Viscosity Viscosity NumberExcipient (mg/mL) (cP) Reduction 17.1 None 0 228.6  0% 17.2Alpha-Cyclodextrin 20 151.5 34% sulfated Na salt 17.3 K acetate 40 89.560%

Example 18: Effect of Excipients on Viscosity of PEGylated BSA withMultiple PEG Chains Per BSA Molecule

A formulation of PEGylated BSA (from Example 15 above) with citric acidNa salt as excipient was prepared by adding 8 milligrams of theexcipient salt to 0.2 mL of the PEGylated BSA solution. The solution wasmixed by gently shaking and the viscosity was measured by a RheoSensemicroVisc equipped with an A10 channel (100 micron depth) at a shearrate of 500 sec-1. The viscometer measurements were completed at ambienttemperature. The results presented in Table 9 shows the effect of theadded excipient compounds in reducing viscosity.

TABLE 9 Excipient Test Concentration Viscosity Viscosity NumberExcipient Added (mg/mL) (cP) Reduction 18.1 None 0 56.8  0% 18.2 Citricacid Na salt 40 43.5 23%

Example 19: Effect of Excipients on Viscosity of PEGylated Lysozyme withMultiple PEG Chains Per Lysozyme Molecule

A formulation of PEGylated lysozyme (from Example 16 above) withpotassium acetate as excipient was prepared by adding 6 milligrams ofthe excipient salt to 0.3 mL of the PEGylated lysozyme solution. Thesolution was mixed by gently shaking and the viscosity was measured by aRheoSense microVisc equipped with an A10 channel (100 micron depth) at ashear rate of 500 sec−1. The viscometer measurements were completed atambient temperature. The results presented in the next table shows thebenefit of the added excipient compounds in reducing viscosity.

TABLE 10 Excipient Test Concentration Viscosity Viscosity NumberExcipient (mg/mL) (cP) Reduction 19.1 None 0 24.6 0% 19.2 K acetate 2022.6 8%

Example 20: Protein Formulations Containing Excipient Combinations

Formulations were prepared using an excipient compound or a combinationof two excipient compounds and a test protein, where the test proteinwas intended to simulate a therapeutic protein that would be used in atherapeutic formulation. These formulations were prepared in 20 mMhistidine buffer with different excipient compounds for viscositymeasurement in the following way. Excipient combinations were dissolvedin 20 mM histidine and the resulting solution pH adjusted with smallamounts of sodium hydroxide or hydrochloric acid to achieve pH 6 priorto dissolution of the model protein. Excipient compounds for thisExample are listed below in Table 11. Once excipient solutions had beenprepared, the test protein bovine gamma globulin (BGG) was dissolved ata ratio to achieve a final protein concentration of about 280 mg/mL.Solutions of BGG in the excipient solutions were formulated in 5 mLsterile polypropylene tubes and allowed to shake at 80-100 rpm on anorbital shaker table overnight. BGG solutions were then transferred to 2mL microcentrifuge tubes and centrifuged for about ten minutes at 2300rpm in an IEC MicroMax microcentrifuge to remove entrained air prior toviscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25° C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation andsubsequent twenty second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, and theresults are shown in Table 11 below. The normalized viscosity is theratio of the viscosity of the model protein solution with excipient tothe viscosity of the model protein solution with no excipient.

TABLE 11 Excipient A Excipient B Conc. Conc. Normalized Test # Name(mg/mL) Name (mg/mL) Viscosity 20.1 None 0 None 0 1.00 20.2 Aspartame 10None 0 0.83 Saccharin 60 None 0 0.51 20.4 Acesulfame K 80 None 0 0.4420.5 Theophylline 10 None 0 0.84 20.6 Saccharin 30 None 0 0.58 20.7Acesulfame K 40 None 0 0.61 20.8 Caffeine 15 Taurine 15 0.82 20.9Caffeine 15 Tyramine 15 0.67

Example 21: Protein Formulations Containing Excipients to ReduceViscosity and Injection Pain

Formulations were prepared using an excipient compound, a secondexcipient compound, and a test protein, where the test protein wasintended to simulate a therapeutic protein that would be used in atherapeutic formulation. The first excipient compound, Excipient A, wasselected from a group of compounds having local anesthetic properties.The first excipient, Excipient A and the second excipient, Excipient Bare listed in Table 12. These formulations were prepared in 20 mMhistidine buffer using Excipient A and Excipient B in the following way,so that their viscosities could be measured. Excipients in the amountsdisclosed in Table 12 were dissolved in 20 mM histidine and theresulting solutions were pH adjusted with small amounts of sodiumhydroxide or hydrochloric acid to achieve pH 6 prior to dissolution ofthe model protein. Once excipient solutions had been prepared, the testprotein bovine gamma globulin (BGG) was dissolved in the excipientsolution at a ratio to achieve a final protein concentration of about280 mg/mL. Solutions of BGG in the excipient solutions were formulatedin 5 mL sterile polypropylene tubes and allowed to shake at 80-100 rpmon an orbital shaker table overnight. BGG-excipient solutions were thentransferred to 2 mL microcentrifuge tubes and centrifuged for about tenminutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of the formulations prepared as described abovewere made with a DV-IIT LV cone and plate viscometer (BrookfieldEngineering, Middleboro, Mass.). The viscometer was equipped with aCP-40 cone and was operated at 3 rpm and 25° C. The formulation wasloaded into the viscometer at a volume of 0.5 mL and allowed to incubateat the given shear rate and temperature for 3 minutes, followed by ameasurement collection period of twenty seconds. This was then followedby 2 additional steps consisting of 1 minute of shear incubation andsubsequent twenty second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient, and theresults are shown in Table 12 below. The normalized viscosity is theratio of the viscosity of the model protein solution with excipient tothe viscosity of the model protein solution with no excipient.

TABLE 12 Excipient A Excipient B Conc. Conc. Normalized Test # Name(mg/mL) Name (mg/mL) Viscosity 21.1 None  0 None  0 1.00 21.2 Lidocaine45 None  0 0.73 21.3 Lidocaine 23 None  0 0.74 21.4 Lidocaine 10Caffeine 15 0.71 21.5 Procaine HCl 40 None  0 0.64 21.6 Procaine HCl 20Caffeine 15 0.69

Example 22: Formulations Containing Excipient Compounds and PEG

Formulations were prepared using an excipient compound and PEG, wherethe PEG was intended to simulate a therapeutic PEGylated protein thatwould be used in a therapeutic formulation, and where the excipientcompounds were provided in the amounts as listed in Table 13. Theseformulations were prepared by mixing equal volumes of a solution of PEGwith a solution of the excipient. Both solutions were prepared indeionized (DI) Water.

The PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide)average Mw 100,000 (Aldrich Catalog #181986) with 83.5 g of DI water.The mixture was stirred overnight for complete dissolution.

The excipient solutions were prepared by this general method and asdetailed in Table 13 below: An approximately 20 mg/mL solution ofpotassium phosphate tribasic (Aldrich Catalog #P5629) in DI water wasprepared by dissolving 0.05 g of potassium phosphate in 5 mL of DIwater. The PEG excipient solution was prepared by mixing 0.5 mL of thePEG solution with 0.5 mL of the excipient solution and mixed by using avortex for a few seconds. A control sample was prepared by mixing 0.5 mLof the PEG solution with 0.5 mL of DI water. Viscosity was measured andresults are recorded in Table 13 below.

TABLE 13 Excipient Viscosity Test Concentration Viscosity ReductionNumber Excipient (mg/mL) (cP) (%) 22.1 None  0 79.7  0   22.2 Citricacid Na salt 10 74.9  6.0 22.3 Potassium phosphate 10 72.3  9.3 22.4Citric acid Na salt/ 10/10 69.1 13.3 Potassium phosphate 22.5 Sodiumsulfate 10 75.1  5.8 22.6 Citric acid Na salt/ 10/10 70.4 11.7 Sodiumsulfate

Example 23: Improved Processing of Protein Solutions with Excipients

Two BGG solutions were prepared by mixing 0.25 g of solid BGG with 4 mlof a buffer solution. For Sample A: Buffer solution was 20 mM histidinebuffer (pH=6.0). For sample B: Buffer solution was 20 mM histidinebuffer containing 15 mg/ml of caffeine (pH=6). The dissolution of thesolid BGG was carried out by placing the samples in an orbital shakerset at 100 rpm. The buffer sample containing caffeine excipient wasobserved to dissolve the protein faster. For the sample with thecaffeine excipient (Sample B) complete dissolution of the BGG wasachieved in 15 minutes. For the sample without the caffeine (Sample A)the dissolution needed 35 minutes.

Next the samples were placed in 2 separate Amicon Ultra 4 CentrifugalFilter Unit with a 30,000 molecular weight cut off and the samples werecentrifuged at 2,500 rpm at 10 minutes intervals. The filtrate volumerecovered after each 10 minute centrifuge run was recorded. The resultsin Table 14 show the faster recovery of the filtrate for Sample B. Inaddition, Sample B kept concentrating with every additional run butSample A reached a maximum concentration point and furthercentrifugation did not result in further sample concentration.

TABLE 14 Sample A Sample B Centrifuge filtrate collected filtratecollected time (min) (mL) (mL)  10 0.28 0.28  20 0.56 0.61  30 0.78 0.88 40 0.99 1.09  50 1.27 1.42  60 1.51 1.71  70 1.64 1.99  80 1.79 2.29 90 1.79 2.39 100 1.79 2.49

Example 24: Protein Formulations Containing Multiple Excipients

This example shows how the combination of caffeine and arginine asexcipients has a beneficial effect on decreasing viscosity of a BGGsolution. Four BGG solutions were prepared by mixing 0.18 g of solid BGGwith 0.5 mL of a 20 mM Histidine buffer at pH 6. Each buffer solutioncontained different excipient or combination of excipients as describedin the table below. The viscosity of the solutions was measured asdescribed in previous examples. The results show that the hindered amineexcipient, caffeine, can be combined with known excipients such asarginine, and the combination has better viscosity reduction propertiesthan the individual excipients by themselves.

TABLE 15 Viscosity Viscosity Reduction Sample Excipient added (cP) (%) ANone 130.6  0 B Caffeine (10 mg/ml)   87.9 33 C Caffeine (10 mg/ml)/ 66.1 49 Arginine (25 mg/ml)  D Arginine (25 mg/ml)   76.7 41

Arginine was added to 280 mg/mL solutions of BGG in histidine buffer atpH 6. At levels above 50 mg/mL, adding more arginine did not decreaseviscosity further, as shown in Table 16.

TABLE 16 Arginine added Viscosity Viscosity (mg/mL) (cP) reduction (%)  0 79.0  0%  53 40.9 48%  79 46.1 42% 105 47.8 40% 132 49.0 38% 15848.0 39% 174 50.3 36% 211 51.4 35%

Caffeine was added to 280 mg/mL solutions of BGG in histidine buffer atpH 6. At levels above 10 mg/ml, adding more caffeine did not decreaseviscosity further, as shown in Table 17.

TABLE 17 Caffeine added Viscosity Viscosity (mg/mL) (cP) reduction (%) 0 79  0% 10 60 31% 15 62 23% 22 50 45%

Example 25: Caffeine Effect During TFF Concentration Process

In this Example, bovine gamma globulin (BGG) solutions were concentratedin the presence and absence of caffeine using tangential flow filtration(TFF). The Labscale TFF System, produced by EMD Millipore (Billerica,Mass.) was used to perform the experiments. The system was fitted with aPellicon XL TFF cassette that contained an Ultracel membrane with 30 kDamolecular weight cutoff (EMD Millipore, Billerica, Mass.). The nominalmembrane surface area was 50 cm². The feed pressure to the cassette wasmaintained at 30 psi while the retentate pressure was maintained at 10psi. The filtrate flux was monitored over the course of the experimentby measuring its mass as a function of time. Approximately 12 grams ofBGG were dissolved into 500 mL of buffer containing 15 mg/mL caffeine,150 mM NaCl, and 20 mM histidine, adjusted to pH 6. A control sample wasprepared by dissolving 12 grams of BGG into 500 mL of buffer containing150 mM NaCl, and 20 mM histidine, adjusted to pH 6. The buffercomponents were purchased from Sigma-Aldrich. Both solutions werefiltered through a 0.2 μm PES filter (VWR, Radnor, Pa.) prior to TFFprocessing. The performance of the test sample and control sample duringTFF were measured by the mass transfer coefficient. The mass transfercoefficient was determined for each sample using the following equation(as described in J. Hung, A. U. Borwankar, B. J. Dear, T. M. Truskett,K. P. Johnston, High concentration tangential flow ultrafiltration ofstable monoclonal antibody solutions with low viscosities. J. Memb. Sci.508, 113-126 (2016)):

J=k _(c) ln(C _(w) /C _(b))  (Eq. 3)

Eq. 3 describes the filtrate flux J, where k_(c) is the mass transfercoefficient, C_(w) is the protein concentration in the vicinity of themembrane, and C_(b) is the concentration in the liquid bulk, and Eq. 3thereby permits calculation of the mass transfer coefficient L. A graphof the calculated flux J against the ln(C_(b)) yields a linear plot withslope of −kc. Here the flux J is calculated by taking the derivative ofthe filtrate mass with respect to time and C_(b) is calculated using amass-balance. The best-fit mass transfer coefficients are listed inTable 18. The introduction of 15 mg/mL caffeine increased the value ofthe mass transfer coefficient by ˜13%, from 22.5 to 25.4 Lm⁻² hr⁻¹(LMH).

TABLE 18 Sample Mass Transfer Coefficient k_(c) (LMH) Control 22.5 ± 0.115 mg/mL caffeine 25.4 ± 0.1

Example 26: Caffeine Effect During TFF Concentration Process

In this Example, bovine gamma globulin (BGG) solutions were concentratedin the presence and absence of caffeine using tangential flow filtration(TFF). The Labscale TFF System, produced by EMD Millipore (Billerica,Mass.) was used to perform the experiments. The system was fitted with aPellicon XL TFF cassette that contained an Ultracel membrane with 30 kDamolecular weight cutoff (EMD Millipore, Billerica, Mass.). The nominalmembrane surface area was 50 cm². A control sample was prepared bydissolving 14.6 grams of BGG into 582 mL of buffer containing 150 mMNaCl, and 20 mM histidine, adjusted to pH 6, such that the initial BGGconcentration was nominally 25.1 mg/mL. The material was filteredthrough a 0.2 μm PES filter (VWR, Radnor, Pa.) and then processed in theTFF device. The pump speed was adjusted such that the feed pressure wasinitially 30 psi and the retentate valve was adjusted such that theretentate pressure was initially 10 psi. The material was concentratedwithout adjusting either the pump speed or retentate valve for 4.1hours. The initial and final concentrations were determined to be25.4±0.6 and 159±6 mg/mL, respectively, by a Bradford assay, as shown inTable 19 below. A caffeine-containing sample was prepared by dissolving14.2 g of BGG into 566 mL of buffer containing 15 mg/mL caffeine, 150 mMNaCl, and 20 mM histidine, adjusted to pH 6, such that the initial BGGconcentration was nominally 25.1 mg/mL. The material was filteredthrough a 0.2 μm PES filter (VWR, Radnor, Pa.) and then processed in theTFF device. The pump speed and retentate valve were set to identicallevels to those previously. The feed and retentate pressures wereconfirmed to be 30 psi and 10 psi, respectively, as previously. Thematerial was concentrated without adjusting either the pump speed orretentate valve for 4.1 hours. The initial and final concentrations weredetermined to be 24.4±0.5 and 225±10 mg/mL, respectively, by a Bradfordassay, as shown in Table 19 below. The use of caffeine during TFFprocessing increased the final protein concentration by approximately42% when compared to the control, from 159 to 225 mg/mL.

TABLE 19 Initial concentration Final concentration Sample (mg/mL)(mg/mL) Control 25.4 ± 0.6 159 ± 6  15 mg/mL caffeine 24.4 ± 0.5 225 ±10

Example 27: Caffeine Effect During Sterile Filtration of BGG Solutions

Bovine gamma globulin (BGG), L-histidine, and caffeine were purchasedfrom Sigma-Aldrich (St. Louis, Mo., product numbers G5009, H6034, andC7731, respectively). Deionized (DI) water was generated from tap waterwith a Direct-Q 3 UV purification system from EMD Millipore (Billerica,Mass.). 25-mm polyethersulfone (PES) filters with 0.2-μm pores werepurchased from GE Healthcare (Chicago, Ill., catalog number 6780-2502).1-mL Luer-Lok syringes were purchased from Becton, Dickinson and Company(Franklin Lakes, N.J., reference number 309628). A 20-mM histidinebuffer, pH 6.0 was prepared using L-histidine, DI water, and titrated topH 6.0 with 1 M HCl. A 15 mg/mL solution of caffeine was prepared usingthe histidine buffer. The caffeine-free and caffeine-containing bufferswere used to reconstitute BGG to a final concentration of about 280mg/mL. The protein concentration, c, was calculated using:

$\begin{matrix}{c = \frac{m_{p}}{b + {vm}_{p}}} & \left( {{Eq}.4} \right)\end{matrix}$

where m_(p) is the protein mass, b is the volume of buffer added, and vis the partial specific volume of BGG, here taken to be 0.74 mL/g. Theviscosity of each sample was measured using microVisc rheometer(RheoSense, San Ramon, Calif.) at a temperature of 23° C. and shear rateof 250 s¹. The energies required to pass the BGG solutions through thesterile filters were measured using a Tensile Compression Tester (TCT,Instron, Needham, Mass., part number 3343) fitted with a 100 N load cell(Instron, Needham, Mass., part number 2519-103). The syringe plungerswere depressed at a rate of 159 mm/min for a distance of 50 mm. Theenergy requirements were calculated by integrating theload-versus-extension curves measured by the TCT, and results aresummarized in Table 20 below.

TABLE 20 Protein Caffeine Energy concentration concentration Viscosityrequirement Sample (mg/mL) (mg/mL) (cP) (mJ) 1 280  0   106   198 2 28015.1  68.9 181

Example 28: Excipients to Improve Protein-A Chromatography Elution

Four purified, research-grade biosimilar antibodies, ipilimumab,ustekinumab, omalizumab, and tocilizumab were purchased from Bioceros(Utrecht, The Netherlands). They were provided as frozen aliquots atprotein concentrations of 20, 26, 15 and 23 mg/mL, respectively, in anaqueous 40 mM sodium acetate, 50 mM tris-HCl buffer at pH 5.5. Theprotein solutions were thawed at room temperature prior to measurementand afterwards, were filtered through a 0.2 μm polyethersulfone filters.The filtered protein stock solutions were mixed in 1:1 ratio of proteinstock solution to a binding buffer. The binding buffer, used to promotethe binding of the antibodies to the Protein-A resin, was composed of0.1 M sodium phosphate and 0.15 sodium chloride at pH 7.2 in deionized(DI) water. The DI water was produced by purifying tap water with aDirect-Q 3 UV purification system from EMD Millipore (Billerica, Mass.).These solutions were employed to perform Protein-A binding and elutionstudies using a PIERCE™ Protein-A Spin Plate for IgG Screening(ThermoFisher Scientific catalog #45202). The plate had 96 wells, eachcontaining 50 μL of Protein-A resin. The resin was washed with bindingbuffer by adding 200 μL of binding buffer to each well and centrifugingthe plate at 1000×g for 1 minute and discarding the flow-through. Allsubsequent centrifugation steps were performed at 1000×g for 1 minute.This wash procedure was repeated once. Following these initial washingsteps, the diluted protein samples, i.e., samples containing ipilimumab,ustekinumab, omalizumab, and tocilizumab, were added to the wells in theplate (200 μL per well). The plate was then placed on a DaiggerScientific (Vernon Hills, Ill.) Labgenius orbital shaker and agitated at260 rpm for 30 minutes, following which the plate was centrifuged andthe flow-through was discarded. The wells were then washed by adding 500μL of binding buffer to each well, centrifuging the plate and discardingthe flow-through. This wash step was repeated twice. After these washingsteps, the proteins were eluted from the plate using elution buffers towhich different excipients had been added. For each elution, 50 μL of aneutralization buffer consisting of 1 M sodium phosphate at pH 7 wasadded to each well of the collection plate, and then two hundred μL ofelution buffer was added to each well of the plate. The plate wasagitated at 260 rpm for 1 minute and then centrifuged. The flow-throughwas recovered for analysis. This elution step was repeated once. Thecontrol buffer, with no excipients, contained 20 mM citrate and had a pHof 2.6. Because Protein-A elution buffers often contain some amount ofsalt, an elution buffer of 100 mM NaCl in the citrate buffer wasprepared as a secondary control.

Table 21 lists the excipient solutions used in this example, theirconcentrations, and final pH of the elution buffers. All excipients werepurchased from Sigma Aldrich (St. Louis, Mo.), with the exception ofaspartame, which was purchased from Herb Store USA (Los Angeles,Calif.), trehalose, which was purchased from Cascade Analytical Reagentsand Biochemicals (Corvallis, Oreg.), and sucrose which was purchasedfrom Research Products International (Mt. Prospect, Ill., product numberS24060). All excipient-containing elution buffers were prepared bymixing the appropriate quantity of the excipient with approximately 10mL of the salt-free citrate buffer control. The elution buffers wereprepared at approximately 100 mM excipient. However, not all of theexcipients are soluble at this level; Table 21 therefore lists all ofthe excipient concentrations that were used. The pH of each elutionbuffer was adjusted to about 2.6±0.1 using either hydrochloride orsodium hydroxide as needed.

For each protein sample, ASD High performance size-exclusionchromatography (SEC) analysis was performed using a TSKgel SuperSW3000column (30 cm×4.6 mm ID, Tosoh Bioscience, King of Prussia, Pa.)connected to an HPLC workstation (Agilent HP 1100 system). Theseparation was carried out at a flow of 0.35 mL/min at room temperature.The mobile phase was an aqueous buffer of 100 mM sodium phosphate, 300mM sodium chloride, pH 7. The protein concentration was monitored byabsorbance at 280 nm using an Agilent 1100 Series G1315B diode arraydetector. The total amount of protein eluted from the Protein-A resinfor each protein, i.e., ipilimumab, ustekinumab, omalizumab, andtocilizumab, was estimated by integrating the chromatograms. Theintegrated peak areas for each protein, i.e., ipilimumab, ustekinumab,omalizumab, and tocilizumab, are listed in Tables 22-25. Tables 22-25also compare the experimental peak areas to those of the salt-free andsalt-containing controls. Values greater than 100% indicate that theelution buffer recovered more protein from the Protein-A resin than thecontrol whereas values less than 100% indicate that the elution bufferrecovered less protein from the Protein-A resin than the control.

TABLE 21 Excipients used in Example 28 Sigma-Aldrich Excipient productnumber concentration Excipient for excipient (mM) pH caffeine C7731  792.6 acesulfame potassium 04054 110 2.5 1-methyl-2-pyrrolidone M6762 1172.6 aspartame N/A  20 2.6 taurine T8691 114 2.5 trehalose N/A 100 2.7sucrose N/A 101 2.7 niacinamide N5535  99 2.7 sodium chloride controlS7653 117 2.6 control N/A N/A 2.5

TABLE 22 Ipilimumab recovery from Protein-A resin Peak area Peak areaPeak area normalized to salt- normalized to Excipient (mAU * min) freecontrol (%) salt control (%) citrate 3409  77.9  83.6 Acesulfame 1567 35.8  38.4 potassium 1-methyl-2-  386   8.8   9.5 pyrrolidone aspartame4012  91.7  98.3 taurine 3958  90.4  97.0 trehalose 3667  83.8  89.9sucrose 4585 104.8 112.4 niacinamide 4295  98.2 105.3 sodium 4080  93.2100.0 chloride control control 4376 100.0 107.2

TABLE 23 Ustekinumab recovery from Protein-A resin Integrated Peak areaPeak area peak area normalized to salt- normalized to Excipient (mAU *min) free control (%) salt control (%) caffeine 2301  86.6  75.2acesulfame  307  16.2  14.0 potassium aspartame  417  17.4  15.11-methyl-2- 2952 108.8  94.4 pyrrolidone taurine 3257 118.6 103.0trehalose 1549  56.6  49.1 sucrose 1274  51.2  44.4 niacinamide 3204116.1 100.8 sodium chloride 3176 115.2 100.0 control

TABLE 24 Omalizumab recovery from Protein-A resin Integrated Peak areaPeak area peak area normalized to salt- normalized to Excipient (mAU *min) free control (%) salt control (%) caffeine 4040 105.5 117.5acesulfame 3620  94.5 105.3 potassium 1-methyl-2- 3334  87.0  97.0pyrrolidone aspartame 3605  94.1 104.8 taurine 4337 113.2 126.1trehalose 3571  93.2 103.8 sucrose 3639  95.0 105.8 niacinamide 4812125.6 139.9 sodium chloride 3439  89.8 100.0 control control 3831 100.0111.4

TABLE 25 Tocilizumab recovery from Protein-A resin Integrated Peak areaPeak area peak area normalized to salt- normalized to Excipient (mAU *min) free control (%) salt control (%) caffeine 3120 111.2 100.3acesulfame 3083 109.9  99.1 potassium 1-methyl-2-  261   9.3   8.4pyrrolidone aspartame  556  19.8  17.9 taurine 3054 108.8  98.2trehalose 2781  99.1  89.4 sucrose 1037  37.0  33.3 niacinamide 2550 90.9  82.0 sodium chloride 3111 110.9 100.0 control control 2806 100.0 90.2

Example 29: Excipients to Improve Protein-A Chromatography Elution

The test proteins used in this Example are identical to those in Example28, i.e., ipilimumab, ustekinumab, omalizumab, and tocilizumab.Protein-A binding and elution studies were performed using an identicalplate to that in Example 28. The methods for loading and eluting theantibodies from the Protein-A plate were identical to those in Example28 with the exception of the elution step. In Example 28, two elutionwashes were performed. However, in this Example, only one wash isperformed. As in Example 28, elution buffers were prepared from a 20 mMcitrate, pH 2.6 control buffer. The elution buffers are listed in Table26 below. All of the excipients were purchased from Sigma-Aldrich (St.Louis, Mo.). The recovered protein was analyzed by HPLC in an identicalfashion to that in Example 28, and results of protein recovery for eachprotein, i.e., ipilimumab, ustekinumab, omalizumab, and tocilizumab, aredocumented in Tables 27-30 below.

TABLE 26 Excipients used in Example 29 Excipient Sigma-Aldrichconcentration Excipient product number (mM) pH Control N/A N/A 2.5sodium chloride control S7653 117 2.6 niacinamide N5535  99 2.7 TaurineT8691 114 2.5 imidazole I5513 100 2.6 4-hydroxybenzesulfonic acid 171506107 2.6 Caffeine C7731  79 2.6

TABLE 27 Ipilimumab recovery from Protein-A resin Peak area Peak areanormalized normalized Peak area to salt-free to salt Excipient (mAU*min)control (%) control (%) control 4841 100.0 88.3  sodium chloride control5485 113.3 100.0  niacinamide 6300 130.1 114.8  taurine 7557 156.1137.8  imidazole 6071 125.4 110.7  4-hydroxybenzesulfonic 5836 120.6106.4  acid caffeine 6051 125.0 110.3 

TABLE 28 Ustekinumab recovery from Protein-A resin Peak area Peak areanormalized normalized Peak area to salt-free to salt Excipient (mAU*min)control (%) control (%) control 4572 100.0  107.9  sodium chloridecontrol 4238 92.7  100.0  niacinamide 5848 127.9  138.0  taurine 4744103.8  112.0  imidazole 4617 101.0  108.9  4-hydroxybenzesulfonic 413290.4  97.5  acid Caffeine 5084 111.2  120.0 

TABLE 29 Omalizumab recovery from Protein-A resin Peak area Peak areanormalized normalized Peak area to salt-free to salt Excipient (mAU*min)control (%) control (%) control 4194 100.0  91.7  sodium chloridecontrol 4574 109.1  100.0  niacinamide 5748 137.0  125.7  taurine 4676111.5  102.2  imidazole 2589 61.7  56.6  4-hydroxybenzesulfonic 319076.1  69.7  acid caffeine 5807 138.5  127.0 

TABLE 30 Tocilizumab recovery from Protein-A resin Peak area Peak areanormalized normalized Peak area to salt-free to salt Excipient (mAU*min)control (%) control (%) control 4667 100.0  97.5  sodium chloridecontrol 4786 102.6  100.0  niacinamide 5225 111.9  109.2  taurine 5396115.6  112.7  imidazole 4754 101.9  99.3  4-hydroxybenzesulfonic 453997.3  94.8  acid caffeine 5656 121.2  118.2 

Example 30: Excipients that Improve Omalizumab Elution from Protein-AChromatography Column

Research-grade omalizumab was purchased from Bioceros (Utrecht, TheNetherlands) and provided frozen at 15 mg/mL in an aqueous 40 mM sodiumacetate, 50 mM tris-HCl buffer, pH 5.5. The protein was thawed at roomtemperature prior to experiments and filtered through a 0.2 μmpolyethersulfone filter. The filtered material was mixed in a 1:1 ratiowith a binding buffer that consisted of 20 mM sodium phosphate, pH 7 inDI water. Tap water was purified with a Direct-Q 3 UV purificationsystem from EMD Millipore (Billerica, Mass.) to produce the DI water.Protein-A purification was performed using a HiTrap Protein-A HP 1 mLcolumn from GE Healthcare (Chicago, Ill., product number 29048576). Foreach experiment, the column was first equilibrated with 10 mL of bindingbuffer. Following equilibration, 30 mg of protein were loaded onto theProtein-A column. The column was then washed with 5 mL of bindingbuffer. After washing the column, bound omalizumab was eluted from thecolumn using fractions of one of the elution buffers listed in Table 31below. The elution buffers were prepared by dissolving the indicatedexcipients in a 20 mM citrate buffer, pH 4.0. All elution buffers wereadjusted to pH 4.0. Five 1-mL fractions were collected. Finally,Protein-A was regenerated by washing the column with 5 mL of 100 mMcitrate, pH 3.0 buffer. The flowrate for each step was 1 mL/min, whichwas maintained by a Fusion 100 infusion pump (Chemyx, Stafford, Tex.).10-mL NormJect Luer Lok syringes were used (Henke Sass Wolf, Tuttlingen,Germany, reference number 4100-000V0).

Elution fractions, E1, E2, E3, E4, and E5, were assayed for totalprotein content by high performance size-exclusion chromatography (SEC)analysis. SEC analysis was performed using a TSKgel SuperSW3000 column(30 cm×4.6 mm ID, Tosoh Bioscience, King of Prussia, Pa.) connected toan HPLC workstation (Agilent HP 1100 system). The separation was carriedout at a flow of 0.35 mL/min at room temperature. The mobile phase wasan aqueous buffer of 100 mM sodium phosphate, 300 mM sodium chloride, pH7. The protein concentration was monitored by absorbance at 280 nm usingan Agilent 1100 Series G1315B diode array detector. The total amount ofprotein eluted from the Protein-A resin was estimated by integrating thechromatograms.

Citrate is a common excipient used in Protein-A chromatography and wastherefore used here as a control. The eluate fractions for the controlsample exhibited insoluble aggregates on storage overnight at 4° C. asevidenced by the formation of a precipitate phase. Therefore, the peakareas reported in Table 31 below represent the total soluble proteinamounts in the eluate fractions. We note that insoluble aggregates wereonly observed in the control sample and none of the other samplesexhibited such aggregates. Peak areas greater than that of the control(using the citrate excipient) indicate that the use of the testexcipient can enable a more efficient separation of protein from thecolumn.

TABLE 31 Omalizumab elution from Protein-A column Total E2 peak E3 peakE4 peak E5 peak peak Elution excipient E1 peak area area area area areaElution concentration area (mAU* (mAU* (mAU* (mAU* (mAU* excipient (mM)(mAU*min) min) min) min) min) min) citrate 103 352 9670 4098 4245 295321318 (control) imidazole 100 236 10224 7373 3894 2620 24348 taurine 125408 17018 7676 3349 2211 30662 niacinamide 102 228 14492 5307 2914 201424955 caffeine 81 617 21965 8069 3301 1911 35863

Example 31: Formulations of BGG with Different Amounts of CaffeineExcipient

Formulations were prepared with different molar concentrations ofcaffeine (at concentrations listed in Table 32 below) and a testprotein, where the test protein was intended to simulate a therapeuticprotein that would be used in a therapeutic formulation. Theformulations for this Example were prepared in 20 mM histidine bufferfor viscosity measurement in the following way. Stock solutions of 0 and80 mM caffeine were prepared in 20 mM histidine and the resultingsolution pH adjusted with small amounts of sodium hydroxide orhydrochloric acid to achieve pH 6 prior to dissolution of the modelprotein. Additional solutions at various caffeine concentrations wereprepared by blending the two stock solutions at various volume ratios,to provide a series of caffeine-containing solutions, at concentrationslisted in Table 32 below. Once these excipient solutions had beenprepared, the test protein bovine gamma globulin (BGG) was dissolvedinto each test solution at a ratio to achieve a final proteinconcentration of about 280 mg/mL by adding 0.7 mL of each excipientsolution to 0.25 g lyophilized BGG powder. The BGG-containing solutionswere formulated in 5 mL sterile polypropylene tubes and allowed to shakeat 100 rpm on an orbital shaker table overnight. These solutions werethen transferred to 2 mL microcentrifuge tubes and centrifuged for aboutfive minutes at 2400 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a microVisc viscometer (RheoSense, San Ramon, Calif.). Theviscometer was equipped with an A-10 chip having a channel depth of 100microns, and was operated at a shear rate of 250 l/s and 25° C. Tomeasure viscosity, the test formulation was loaded into the viscometer,taking care to remove all air bubbles from the pipet. The pipetcontaining the loaded sample formulation was placed in the instrumentand allowed to incubate at the measurement temperature for about fiveminutes. The instrument was then run until the channel was fullyequilibrated with the test fluid, indicated by a stable viscosityreading, and then the viscosity recorded in centipoise. Viscosityresults that were obtained are presented in Table 32 below.

TABLE 32 Caffeine Viscosity Normalized conc (mM) (cP) Viscosity  0 831.00  5 67 0.81 10 70 0.84 20 77 0.92 30 63 0.76 40 65 0.78 50 65 0.7860 57 0.69 70 50 0.60 80 50 0.60

Example 32: Preparation of Solutions of Co-Solutes in Deionized Water

Compounds used as co-solutes to increase caffeine solubility in waterwere obtained from Sigma-Aldrich (St. Louis, Mo.) and includedniacinamide, proline, procaine HCl, ascorbic acid, 2,5-dihydroxybenzoicacid, lidocaine, saccharin, acesulfame K, tyramine, and aminobenzoicacid. Solutions of each co-solute were prepared by dissolving dry solidin deionized water and in some cases adjusting the pH to a value betweenpH of about 6 and pH of about 8 with 5 M hydrochloric acid or 5 M sodiumhydroxide as necessary. Solutions were then diluted to a final volume ofeither 25 mL or 50 mL using a Class A volumetric flask and concentrationrecorded based on the mass of compound dissolved and the final volume ofthe solution. Prepared solutions were used either neat or diluted withdeionized water.

Example 33: Caffeine Solubility Testing

The impact of different co-solutes on the solubility of caffeine atambient temperature (about 23° C.) was assessed in the following way.Dry caffeine powder (Sigma-Aldrich, St. Louis, Mo.) was added to 20 mLglass scintillation vials and the mass of caffeine recorded. 10 mL of aco-solute solution prepared in accordance with Example 32 was added tothe caffeine powder in certain cases; in other cases, a blend of aco-solute solution and deionized water was added to the caffeine powder,maintaining a final addition volume of 10 mL. The volume contribution ofthe dry caffeine powder was assumed to be negligible in any of thesemixtures. A small magnetic stir bar was added to the vial, and thesolution was allowed to mix vigorously on a stir plate for about 10minutes. After about 10 minutes the vial was observed for dissolution ofthe dry caffeine powder, and the results are given in Table 33 below.These observations indicated that niacinamide, procaine HCl,2,5-dihydroxybenzoic acid sodium salt, saccharin sodium salt, andtyramine chloride salt all enabled dissolution of caffeine to at leastabout four times the reported caffeine solubility limit (˜16 mg/mL atroom temperature according to Sigma-Aldrich).

TABLE 33 Co-solute Conc. Caffeine Test No. Name (mg/mL) (mg/mL)Observation 33.1  Proline 100  50 DND 33.2  Niacinamide 100  50   CD33.3  Niacinamide 100  60   CD 33.4  Niacinamide 100  75   CD 33.5 Niacinamide 100  85   CD 33.6  Niacinamide 100 100   CD 33.7 Niacinamide  80  85   CD 33.8  Niacinamide  50  80   CD 33.9  ProcaineHCl 100  85   CD 33.10 Procaine HCl  50  80   CD 33.11 Niacinamide  30 80 DND 33.12 Procaine HCl  30  80 DND 33.13 Niacinamide  40  80   MD33.14 Procaine HCl  40  80 DND 33.15 Ascorbic acid, Na  50  80 DND 33.16Ascorbic acid, Na 100  80 DND 33.17 2,5 DHBA, Na  40  80   CD 33.18 2,5DHBA, Na  20  80   MD 33.19 Lidocaine HCl  40  80 DND 33.20 Saccharin,Na  90  80   CD 33.21 Acesulfame K  80  80 DND 33.22 Tyramine HCl  60 80   CD 33.23 Na Aminobenzoate  46  80 DND 33.24 Saccharin, Na  45  80DND 33.25 Tyramine HCl  30  80 DND CD = completely dissolved; MD =mostly dissolved; DND = did not dissolve

Example 34: Profile of HUMIRA®

HUMIRA® (AbbVie Inc., Chicago, Ill.) is a commercially availableformulation of the therapeutic monoclonal antibody adalimumab, aTNF-alpha blocker typically prescribed to reduce inflammatory responsesof autoimmune diseases such as rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,moderate to severe chronic psoriasis and juvenile idiopathic arthritis.HUMIRA® is sold in 0.8 mL single use doses containing 40 mg ofadalimumab, 4.93 mg sodium chloride, 0.69 mg sodium phosphate monobasicdihydrate, 1.22 mg sodium phosphate dibasic dihydrate, 0.24 mg sodiumcitrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol and 0.8 mgpolysorbate 80. A viscosity vs. concentration profile of thisformulation was generated in the following way. An Amicon Ultra 15centrifugal concentrator with a 30 kDa molecular weight cut-off(EMD-Millipore, Billerica, Mass.) was filled with about 15 mL ofdeionized water and centrifuged in a Sorvall Legend RT (ThermoFisherScientific) at 4000 rpm for 10 minutes to rinse the membrane. Afterwardsthe residual water was removed and 2.4 mL of HUIMIRA® liquid formulationwas added to the concentrator tube and was centrifuged at 4000 rpm for60 minutes at 25° C. Concentration of the retentate was determined bydiluting 10 microliters of retentate with 1990 microliters of deionizedwater, measuring absorbance of the diluted sample at 280 nm, andcalculating the concentration using the dilution factor and extinctioncoefficient of 1.39 mL/mg-cm. Viscosity of the concentrated sample wasmeasured with a microVisc viscometer equipped with an A05 chip(RheoSense, San Ramon, Calif.) at a shear rate of 250 sec⁻¹ at 23° C.After viscosity measurement, the sample was diluted with a small amountof filtrate and concentration and viscosity measurements were repeated.This process was used to generate viscosity values at varying adalimumabconcentrations, as set forth in Table 34 below.

TABLE 34 Adalimumab concentration Viscosity (mg/mL) (cP) 277 125 253  63223  34 202  20 182  13

Example 35: Reformulation of HUMIRA® with Viscosity-Reducing Excipient

The following example describes a general process by which HUMIRA® wasreformulated in buffer with viscosity-reducing excipient. A solution ofthe viscosity-reducing excipient was prepared in 20 mM histidine bydissolving about 0.15 g histidine and 0.75 g caffeine (Sigma-Aldrich,St. Louis, Mo.) in deionized water. The pH of the resulting solution wasadjusted to about 5 with 5 M hydrochloric acid. The solution was thendiluted to a final volume of 50 mL in a volumetric flask with deionizedwater. The resulting buffered viscosity-reducing excipient solution wasthen used to reformulate HUMIRA® at high mAb concentrations. Next, about0.8 mL of HUMIRA® was added to a rinsed Amicon Ultra 15 centrifugalconcentrator tube with a 30 kDa molecular weight cutoff and centrifugedin a Sorvall Legend RT at 4000 rpm and 25° C. for 8-10 minutes.Afterwards about 14 mL of the buffered viscosity-reducing excipientsolution prepared as described above was added to the concentratedHUMIRA® in the centrifugal concentrator. After gentle mixing, the samplewas centrifuged at 4000 rpm and 25° C. for about 40-60 minutes. Theretentate was a concentrated sample of HUMIRA® reformulated in a bufferwith viscosity-reducing excipient. Viscosity and concentration of thesample were measured, and in some cases then diluted with a small amountof filtrate to measure viscosity at a lower concentration. Viscositymeasurements were completed with a microVisc viscometer in the same wayas with the concentrated HUMIRA® formulation in the previous example.Concentrations were determined with a Bradford assay using a standardcurve generated from HUMIRA® stock solution diluted in deionized water.Reformulation of HUMIRA® with the viscosity-reducing excipient gaveviscosity reductions of 30% to 60% compared to the viscosity values ofHUMIRA® concentrated in the commercial buffer without reformulation, asset forth in Table 35 below.

TABLE 35 Adalimumab concentration Viscosity (mg/mL) (cP) 290 61 273 48244 20 205 14

Example 36: Improved Stability of Adalimumab Solutions with Caffeine asExcipient

The stability of adalimumab solutions with and without caffeineexcipient was evaluated after exposing samples to 2 different stressconditions: agitation and freeze-thaw. The adalimumab drug formulationHUMIRA® (AbbVie) was used, having properties described in more detail inExample 34. The HUMIRA® sample was concentrated to 200 mg/mL adalimumabconcentration in the original buffer solution as described in Example39; this concentrated sample is designated “Sample 1.” A second samplewas prepared with ˜200 mg/mL of adalimumab and 15 mg/mL of addedcaffeine as described in Example 40; this concentrated sample with addedcaffeine is designated “Sample 2.” Both samples were diluted to a finalconcentration of 1 mg/mL adalimumab with the diluents as follows: Sample1 diluent is the original buffer solution, and Sample 2 diluent is a 20mM histidine, 15 mg/mL caffeine, pH=5. Both HUMIRA® dilutions werefiltered through a 0.22 μm syringe filter. For every diluted sample, 3batches of 300 μL each were prepared in a 2 mL Eppendorf tube in alaminar flow hood. The samples were submitted to the following stressconditions: for agitation, samples were placed in an orbital shaker at300 rpm for 91 hours; for freeze-thaw, samples were cycled 7 times from−17 to 30° C. for an average of 6 hours per condition. Table 36describes the samples prepared.

TABLE 36 Sample # Excipient added Stress condition 1-C  None None 1-A None Agitation 1-FT None Freeze-Thaw 2-C  15 mg/mL caffeine None 2-A  15mg/mL caffeine Agitation 2-FT 15 mg/mL caffeine Freeze-Thaw

Example 37: Evaluation of Stability by Dynamic Light Scattering (DLS)

A Brookhaven Zeta Plus dynamic light scattering instrument was used tomeasure the hydrodynamic radius of the adalimumab molecules in thesamples from Example 36, and to look for evidence of the formation ofaggregate populations. Table 37 shows the DLS results for the 6 samplesprepared according to Example 36: some of them (1-A, 1-FT, 2-A, and2-FT) had been exposed to stress conditions (“Stressed Samples”), andothers (1-C and 2-C) had not been stressed. The DLS data in Table 37show a multimodal particle size distribution of the monoclonal antibodyin Stressed Samples that do not contain caffeine. In the absence ofcaffeine as an excipient, the Stressed Samples 1-A and 1-FT showedhigher effective diameter than non-stressed Sample 1-C, and in additionthey showed a second population of particles of significantly higherdiameter; this new grouping of particles with a larger diameter isevidence of aggregation into subvisible particles. The Stressed Samplescontaining the caffeine (Samples 2-A and 2-FT) only display onepopulation of particles, at a particle diameter similar to theunstressed Sample 2-C. These results demonstrate that adding caffeine tothese samples reduced the formation of aggregates or subvisibleparticles.

TABLE 37 Effective Diameter of Diameter of Diameter Population % byIntensity Population % by Intensity Sample # (nm) #1 (nm) of Population#1 #2 (nm) of Population #2 1-C  10.9 10.8 100 — — 1-A  11.5 10.8  8728.9  13 1-FT 20.4 11.5  66 112.2  44 2-C  10.5 10.5 100 — — 2-A  10.810.8 100 — — 2-FT 11.4 11.4 100 — —

Tables 38A and Table 38B display the DLS raw data of adalimumab samplesfrom Example 36 showing the particle size distributions. In theseTables, G(d) is the intensity-weighted differential size distribution.C(d) is the cumulative intensity-weighted differential sizedistribution.

TABLE 38A Sample 1-C Sample 1-A Sample 1-FT Diameter Diameter Diameter(nm) G (d) C(d) (nm) G (d) C(d) (nm) G (d) C(d) 10.6 14 4 9.3 13 3 8.212 2 10.6 53 20 9.8 47 15 9.2 55 13 10.7 92 46 10.3 87 37 10.3 98 3210.8 100 76 10.8 100 63 11.5 100 52 10.9 61 93 11.4 67 80 12.9 57 6310.9 22 100 12 27 87 14.5 14 66 26.1 4 88 89.3 5 67 27.5 10 91 100.1 2772 28.9 13 94 112.2 52 83 30.5 13 97 125.7 52 93 32.1 7 99 140.8 30 9933.8 4 100 157.8 7 100

TABLE 38B Sample 2-C Sample 2-A Sample 2-FT Diameter Diameter Diameter(nm) G (d) C(d) (nm) G (d) C(d) (nm) G (d) C(d) 10.3 14 4 10.6 7 2 11.328 9 10.4 52 19 10.6 43 16 11.3 64 29 10.5 91 46 10.7 79 40 11.4 100 6010.5 100 75 10.8 100 71 11.5 79 85 10.6 62 93 10.8 64 91 11.5 43 98 10.723 100 10.9 29 100 11.6 7 100

Example 38: Evaluation of Stability by Size-Exclusion Chromatography(SEC)

Size exclusion chromatography was used to detect subvisible particulatesof less than about 0.1 microns in size from the stressed and unstressedadalimumab samples described in Example 36. To perform the SEC, a TSKgelSuperSW3000 column (Tosoh Biosciences, Montgomeryville, Pa.) with aguard column was used, and the elution was monitored at 280 nm. A totalof 10 μL of each stressed and unstressed sample from Example 36 waseluted isocratically with a pH 6.2 buffer (100 mM phosphate, 325 mMNaCl), at a flow rate of 0.35 mL/min. The retention time of theadalimumab monomer was approximately 9 minutes. No detectable aggregateswere identified in the samples containing the caffeine excipient, andthe amount of monomer in all 3 samples remained constant.

Example 39: Viscosity Reduction of HERCEPTIN® Formulation

The monoclonal antibody trastuzumab (HERCEPTIN® from Genentech) wasreceived as a lyophilized powder and reconstituted to 21 mg/mL in DIwater. The resulting solution was concentrated as-is in an Amicon Ultra4 centrifugal concentrator tube (molecular weight cut-off, 30 kDa) bycentrifuging at 3500 rpm for 1.5 hrs. The concentration was measured bydiluting the sample 200 times in an appropriate buffer and measuringabsorbance at 280 nm using the extinction coefficient of 1.48 mL/mg.Viscosity was measured using a RheoSense microVisc viscometer.

Excipient buffers were prepared containing salicylic acid and caffeineeither alone or in combination by dissolving histidine and excipients indistilled water, then adjusting pH to the appropriate level. Theconditions of Buffer Systems 1 and 2 are summarized in Table 39.

TABLE 39 Buffer Salicylic Acid Caffeine Osmolality System #concentration concentration (mOsm/kg) pH 1 10 mg/mL 10 mg/mL 145 6 2 015 mg/mL  86 6

HERCEPTIN® solutions were diluted in the excipient buffers at a ratio of˜1:10 and concentrated in Amicon Ultra 15 (MWCO 30 kDa) concentratortubes. Concentration was determined using a Bradford assay and comparedwith a standard calibration curve made from the stock HERCEPTIN® sample.Viscosity was measured using the RheoSense microVisc viscometer. Theconcentration and viscosity measurements of the various HERCEPTIN®solutions are shown in Table 40 below, where Buffer Systems 1 and 2refer to those buffers described in Table 39.

TABLE 40 Buffer System 1: Solution Control solution with with 10 mg/mLCaffeine + Buffer System 2: Solution no added excipients 10 mg/mlSalicylic Acid added with 15 mg/mL Caffeine added Antibody AntibodyAntibody Viscosity Concentration Viscosity Concentration ViscosityConcentration (cP) (mg/mL) (cP) (mg/mL) (cP) (mg/mL) 37.2  215 9.7 24423.4  236 9.3 161 7.7 167 12.2  200 3.1 108 2.9 122 5.1 134 1.6  54 2.4 77 2.1 101

Buffer System 1, containing both salicylic acid and caffeine, had amaximum viscosity reduction of 76% at 215 mg/mL compared to the controlsample. Buffer System 2, containing just caffeine, had viscosityreduction up to 59% at 200 mg/mL.

Example 40: Viscosity Reduction of AVASTIN® Formulation

AVASTIN® (monoclonal antibody bevacizumab formulation marketed byGenentech) was received as a 25 mg/mL solution in a histidine buffer.The sample was concentrated in Amicon Ultra 4 centrifugal concentratortubes (MWCO 30 kDa) at 3500 rpm. Viscosity was measured by RheoSensemicroVisc and concentration was determined by absorbance at 280 nm(extinction coefficient, 1.605 mL/mg). The excipient buffer was preparedby adding 10 mg/mL caffeine along with 25 mM histidine HCl. AVASTIN®stock solution was diluted with the excipient buffer then concentratedin Amicon Ultra 15 centrifugal concentrator tubes (MWCO 30 kDa). Theconcentration of the excipient samples was determined by Bradford assayand the viscosity was measured using the RheoSense microVisc. Resultsare shown in Table 41 below.

TABLE 41 Viscosity with % Viscosity Viscosity 10 mg/mL ReductionConcentration without added added caffeine from (mg/mL) excipient (cP)excipient (cP) Excipient 266 297 113 62% 213  80  22 73% 190  21  13 36%

AVASTIN® showed a maximum viscosity reduction of 73% when concentratedwith 10 mg/mL of caffeine to 213 mg/mL when compared to the controlAVASTIN® sample.

Example 41: Preparation of Formulations Containing Caffeine, a SecondaryExcipient and Test Protein

Formulations were prepared using caffeine as the excipient compound or acombination of caffeine and a second excipient compound, and a testprotein, where the test protein was intended to simulate a therapeuticprotein that would be used in a therapeutic formulation. Suchformulations were prepared in 20 mM histidine buffer with differentexcipient compounds for viscosity measurement in the following way.Excipient combinations (Excipients A and B, as described in Table 28below) were dissolved in 20 mM histidine and the resulting solution pHadjusted with small amounts of sodium hydroxide or hydrochloric acid toachieve pH 6 prior to dissolution of the model protein. Once excipientsolutions had been prepared, the test protein bovine gamma globulin(BGG) was dissolved at a ratio to achieve a final protein concentrationof about 280 mg/mL. Solutions of BGG in the excipient solutions wereformulated in 20 mL glass scintillation vials and allowed to shake at80-100 rpm on an orbital shaker table overnight. BGG solutions were thentransferred to 2 mL microcentrifuge tubes and centrifuged for about tenminutes at 2300 rpm in an IEC MicroMax microcentrifuge to removeentrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25° C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation andsubsequent twenty second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample in Table 42 below. Viscosities of solutions with excipientwere normalized to the viscosity of the model protein solution withoutexcipient. The normalized viscosity is the ratio of the viscosity of themodel protein solution with excipient to the viscosity of the modelprotein solution with no excipient.

TABLE 42 Excipient A Excipient B Conc. Conc. Normalized Name (mg/mL)Name (mg/mL) Viscosity —  0 —  0 1.00 Caffeine 15 —  0 0.77 Caffeine 15Sodium acetate 12 0.77 Caffeine 15 Sodium sulfate 14 0.78 Caffeine 15Aspartic acid 20 0.73 Caffeine 15 CaCl₂ 15 0.65 dihydrate Caffeine 15Dimethyl 25 0.65 Sulfone Caffeine 15 Arginine 20 0.63 Caffeine 15Leucine 20 0.69 Caffeine 15 Phenylalanine 20 0.60 Caffeine 15Niacinamide 15 0.63 Caffeine 15 Ethanol 22 0.65

Example 42: Preparation of Formulations Containing Dimethyl Sulfone andTest Protein

Formulations were prepared using dimethyl sulfone (Jarrow Formulas, LosAngeles, Calif.) as the excipient compound and a test protein, where thetest protein was intended to simulate a therapeutic protein that wouldbe used in a therapeutic formulation. Such formulations were prepared in20 mM histidine buffer for viscosity measurement in the following way.Dimethyl sulfone was dissolved in 20 mM histidine and the resultingsolution pH adjusted with small amounts of sodium hydroxide orhydrochloric acid to achieve pH 6 and then filtered through a 0.22micron filter prior to dissolution of the model protein. Once excipientsolutions had been prepared, the test protein bovine gamma globulin(BGG) was dissolved at a concentration of about 280 mg/mL. Solutions ofBGG in the excipient solutions were formulated in 20 mL glassscintillation vials and allowed to shake at 80-100 rpm on an orbitalshaker table overnight. BGG solutions were then transferred to 2 mLmicrocentrifuge tubes and centrifuged for about ten minutes at 2300 rpmin an IEC MicroMax microcentrifuge to remove entrained air prior toviscosity measurement.

Viscosity measurements of formulations prepared as described above weremade with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.). The viscometer was equipped with a CP-40 cone andwas operated at 3 rpm and 25° C. The formulation was loaded into theviscometer at a volume of 0.5 mL and allowed to incubate at the givenshear rate and temperature for 3 minutes, followed by a measurementcollection period of twenty seconds. This was then followed by 2additional steps consisting of 1 minute of shear incubation andsubsequent twenty second measurement collection period. The three datapoints collected were then averaged and recorded as the viscosity forthe sample. Viscosities of solutions with excipient were normalized tothe viscosity of the model protein solution without excipient. Thenormalized viscosity recorded in Table 43 is the ratio of the viscosityof the model protein solution with excipient to the viscosity of themodel protein solution with no excipient.

TABLE 43 Dimethyl sulfone concentration Normalized (mg/mL) viscosity  01.00 15 0.92 30 0.71 50 0.71 30 0.72

EQUIVALENTS

While specific embodiments of the subject invention have been disclosedherein, the above specification is illustrative and not restrictive.While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. Many variations of the inventionwill become apparent to those of skilled art upon review of thisspecification. Unless otherwise indicated, all numbers expressingreaction conditions, quantities of ingredients, and so forth, as used inthis specification and the claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth herein areapproximations that can vary depending upon the desired propertiessought to be obtained by the present invention.

1. A method of improving a parameter of a protein-related process,comprising: providing a viscosity-reducing excipient additive comprisingat least one excipient compound selected from the group consisting ofhindered amines, anionic aromatics, functionalized amino acids,oligopeptides, short-chain organic acids, low molecular weight aliphaticpolyacids, and diones and sulfones, and adding a viscosity-reducingamount of the at least one excipient compound to a carrier solution forthe protein-related process, wherein the carrier solution contains aprotein of interest, thereby improving the parameter.
 2. The method ofclaim 1, wherein the parameter is selected from the group consisting ofcost of protein production, amount of protein production, rate ofprotein production, and efficiency of protein production.
 3. (canceled)4. The method of claim 1, wherein the protein-related process is anupstream processing process.
 5. The method of claim 4, wherein thecarrier solution for the upstream processing process is a cell culturemedium.
 6. The method of claim 5, wherein the step of adding theexcipient additive to the carrier solution comprises a first substep ofadding the excipient additive to a supplemental medium to form anexcipient-containing supplemental medium, and a second substep of addingthe excipient-containing supplemental medium to the cell culture medium.7. The method of claim 1, wherein the protein-related process is adownstream processing process.
 8. The method of claim 7, wherein thedownstream processing process is a chromatography process.
 9. The methodof claim 8, wherein the chromatography process is a Protein-Achromatography process.
 10. The method of claim 8, wherein thechromatography process recovers the protein of interest, and wherein theprotein of interest is characterized by an improved protein-relatedparameter selected from the group consisting of improved purity,improved yield, fewer particles, less misfolding, or less aggregation,as compared to a control solution.
 11. (canceled)
 12. The method ofclaim 1, wherein the protein-related process is a process selected fromthe group consisting of filtration, injection, syringing, pumping,mixing, centrifugation, membrane separation, and lyophilization.
 13. Themethod of claim 12, wherein the process requires less force than acontrol process.
 14. The method of claim 1, wherein the protein-relatedprocess is selected from the group consisting of a cell culture process,a cell culture harvesting process, a chromatography process, a viralinactivation process, and a filtration process.
 15. The method of claim14, wherein the protein-related process is the viral inactivationprocess, and the viral inactivation process is conducted at a pH levelof about 2.5 to about 5.0.
 16. The method of claim 15, wherein theprotein-related process is the viral inactivation process, and the viralinactivation process is conducted at a higher pH than the controlprocess.
 17. The method of claim 14, wherein the protein-related processis the filtration process.
 18. The method of claim 17, wherein thefiltration process is a virus removal filtration process or anultrafiltration/diafiltration process.
 19. The method of claim 17,wherein the filtration process is characterized by an improvedfiltration-related parameter.
 20. The method of claim 19, wherein theimproved filtration-related parameter is a faster filtration rate thanthe filtration rate of the control solution, production of a smalleramount of aggregated protein than the amount of aggregated proteinproduced by a control filtration process, a higher mass transferefficiency than the mass transfer efficiency of the control filtrationprocess, or a higher concentration or a higher yield of the targetprotein than a concentration or yield of the target protein produced bythe control filtration process. 21-23. (canceled)
 24. The method ofclaim 1, wherein the viscosity-reducing excipient additive comprises twoor more excipient compounds.
 25. The method of claim 1, wherein the atleast one excipient compound is a hindered amine.
 26. The method ofclaim 25, wherein the at least one excipient compound is selected fromthe group consisting of caffeine, saccharin, acesulfame potassium,aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone,2-pyrrolidinone, niacinamide, and imidazole.
 27. The method of claim 26,wherein the at least one excipient compound is selected from the groupconsisting of caffeine, taurine, niacinamide, and imidazole.
 28. Themethod of claim 1, wherein the at least one excipient compound is ananionic aromatic excipient.
 29. (canceled)
 30. The method of claim 1,wherein the viscosity-reducing amount is between about 1 mg/mL to about100 mg/mL of the at least one excipient compound.
 31. The method ofclaim 1, wherein the viscosity-reducing amount is between about 1 mM toabout 400 mM of the at least one excipient compound.
 32. (canceled) 33.The method of claim 1, wherein the carrier solution comprises anadditional agent selected from the group consisting of preservatives,sugars, polysaccharides, arginine, proline, surfactants, stabilizers,and buffers.
 34. The method of claim 1, wherein the protein of interestis a therapeutic protein.
 35. The method of claim 34, wherein thetherapeutic protein is selected from the group consisting of amonoclonal antibody, a polyclonal antibody, an antibody fragment, afusion protein, a PEGylated protein, an antibody-drug conjugate, asynthetic polypeptide, a protein fragment, a lipoprotein, an enzyme, anda structural peptide.
 36. The method of claim 34, wherein thetherapeutic protein is a recombinant protein. 37-39. (canceled)